Method of preparing an undifferentiated cell

ABSTRACT

A method of preparing an undifferentiated cell is described. The method comprises contacting a more committed cell with an agent that causes the more committed cell to retrodifferentiate into an undifferentiated cell.

[0001] The present invention relates to a method of preparing anundifferentiated cell.

[0002] In particular, the present invention relates to a method ofpreparing an undifferentiated cell from a more committed cell.

[0003] In addition the present invention relates to the use of theundifferentiated cell of the present invention for the preparation of anew more committed cell—i.e. a recommitted cell.

[0004] The present invention also relates to the use of theundifferentiated cell of the present invention or the recommitted cellof the present invention to have an effect (directly or indirectly viathe use of products obtained therefrom) on the immune system, such asthe alleviation of symptoms associated with, or the partial or completecure from, an immunological condition or disease.

[0005] By way of introduction, differentiation is a process wherebystructures and functions of cells are progressively committed to giverise to more specialised cells, such as the formation of T cells or Bcells. Therefore, as the cells become more committed, they become morespecialised.

[0006] In contrast, retro-differentiation is a process wherebystructures and functions of cells are progressively changed to give riseto less specialised cells.

[0007] Undifferentiated cells are capable of multilineagedifferentiation—i.e. they are capable of differentiating into two ormore types of specialised cells. A typical example of anundifferentiated cell is a stem cell.

[0008] In contrast, differentiated cells are incapable of multilineagedifferentiation. A typical example of a differentiated cell is a T cell.

[0009] There are many undifferentiated cells and differentiated cellsfound in vivo and the general art is replete with general teachings onthem.

[0010] By way of example, reference may be made to inter alia Levitt andMertelsman 1995 (Haematopoietic Stem Cells, published by Marcel DekkerInc—especially pages 45-59) and Roitt et al (Immunology, 4th Edition,Eds. Roitt, Brostoff and Male 1996, Publ. Mosby—especially Chapter 10).

[0011] In short, however, examples of undifferentiated cells includelymphohaematopoietic progenitor cells (LPCs). LPCs include pluripotentstem cells (PSCs), lymphoid stem cells (LSCs) and myeloid stem cells(MSCs). LSCs and MSCs are each formed by the differentiation of PSCs.Hence, LSCs and MSCs are more committed than PSCs.

[0012] Examples of differentiated cells include T cells, B cells,eosinophils, basophils, neutrophils, megakaryocytes, monocytes,erythrocytes, granulocytes, mast cells, and lymphocytes.

[0013] T cells and B cells are formed by the differentiation of LSCs.Hence, T cells and B cells are more committed than LSCs.

[0014] Eosinophils, basophils, neutrophils, megakaryocytes, monocytes,erythrocytes, granulocytes, mast cells, NKs, and lymphocytes are formedby the differentiation of MSCs. Hence, each of these cells are morecommitted than MSCs.

[0015] Antigens are associated with undifferentiated and differentiatedcells. The term “associated” here means the cells expressing or capableof expressing, or presenting or capable of being induced to present, orcomprising, the respective antigen(s).

[0016] Most undifferentiated cells and differentiated cells compriseMajor Histocompatability Complex (MHC) Class I antigens and/or Class IIantigens. If these antigens are associated with those cells then theyare called Class I⁺ and/or Class II⁺ cells.

[0017] Each specific antigen associated with an undifferentiated cell ora differentiated cell can act as a marker. Hence, different types ofcells can be distinguished from each other on the basis of theirassociated particular antigen(s) or on the basis of a particularcombination of associated antigens.

[0018] Examples of these marker antigens include the antigens CD34, CD19and CD3. If these anigens are present then these particular cells arecalled CD34⁺, CD19³⁰ and CD3³⁰ cells respectively. If these antigens arenot present then these cells are called CD34³¹, CD19³¹ and CD3³¹ cellsrespectively.

[0019] In more detail, PSCs are CD34⁺ cells. LSCs are DR⁺, CD34⁺ andTdT⁺ cells. MSCs are CD34⁺, DR ⁺, CD13⁺, CD33⁺, CD7 ⁺ and TdT⁺ cells. Bcells are CD19 ⁺, CD21³¹, CD22⁺ and DR⁺ cells. T cells are CD2⁺, CD3⁺,and either CD4⁺ or CD8⁺ cells. Immature lymphocytes are CD4⁺ and CD8⁺cells. Activated T cells are DR⁺ cells. Natural killer cells (NKs) areCD56⁺ and CD16⁺ cells. T lymphocytes are CD7⁺ cells. Leukocytes areCD45⁺ cells. Granulocytes are CD13⁺ and CD33⁺ cells. Monocyte macrophagecells are CD14⁺ and DR⁺ cells

[0020] Hence, by looking for the presence of the above-listed antigenmarkers it is possible to identify certain cell types (e.g. whether ornot a cell is an undifferentiated cell or a differentiated cell) and thespecialisation of that cell type (e.g. whether that cell is a T cell ora B cell).

[0021] The general concept of retrodifferentiation is not new. In fact,in 1976 Jose Uriel (Cancer Research 36, 4269-4275. November 1976)presented a review on this topic, in which he said:

[0022] “retrodifferentiation appears as a common adaptive process forthe maintenance of cell integrity against deleterious agents of variedetiology (physical, chemical, and viral). While preserving the entireinformation encoded on its genome, cells undergoing retrodifferentiationlose morphological and functional complexity by virtue of a process ofself-deletion of cytoplasmic structures and the transition to a morejuvenile pattern of gene expression. This results in a progressiveiniformization of originally distinct cell phenotypes and to a decreaseof responsiveness to regulatory signals operational in adult cells.Retrodifferentiation is normally counterbalanced by a process ofreontogeny that tends to restore the terminal phenotypes where thereversion started. This explains why retrodifferentiation remainsinvariably associated to cell regeneration and tissue repair.”

[0023] Uriel (ibid) then went on to discuss cases of reportedretrodifferentiation—such as the work of Gurdon relating to nuclei fromgut epithelial cells of Xenopus tadpoles (Advances in Morphogenesis[1966] vol 4, pp 1-43. New York Academic Press, Eds Abercrombie andBracher), and the work of Bresnick relating to regeneration of liver(Methods in Cancer Research [1971] vol 6, pp 347-391).

[0024] Uriel (ibid) also reported on work relating to isolated liverparenchymal cells for in vitro cultures. According to Uriel:

[0025] “Contrary to the results with fetal or neonatal hepatocytes, withhepatocytes from regenerating liver, or from established hepatomas, ithas been difficult to obtain permanent class lines from resting adulthepatocytes.”

[0026] Uriel (ibid) also reported on apparent retrodifferentiation incancer, wherein he stated:

[0027] “the biochemical phenotypes of many tumours show analogouschanges of reversion toward immaturity . . . during the preneoplasticphase of liver carcinogenesis, cells also retrodifferentiate.”

[0028] More recent findings on retrodifferentiation include the work ofMinoru Fukunda (Cancer Research [1981] vol 41, pp 4621-4628). Fukundainduced specific changes in the cell surface glycoprotein profile ofK562 human leukaemic cells by use of the tumour-promoting phorbol ester,12-O-tetradecanoyl-phorbol-13-acetate (TPA). According to Fukunda TPAappeared to induce the K562 human leukaemic cells into aretrodifferentiated stage.

[0029] Also, Hass et al (Cell Growth & Differentiation [1991 ] vol 2, pp541-548) reported that long term culture of TPA-differentiated U-937leukaemia cells in the absence of phorbol ester for 32-36 days resultedin a process of retrodifferentiation and that the retrodifferentiatedcells detached from the substrate and reinitiated Proliferation.

[0030] Another case of retrodifferentiation is the work of Curtin andSnell (Br. J. Cancer [1983] vol 48, pp 495-505]. These workers comparedenzymatic changes occurring during diethylnitrosamine-inducedhepatocarcinogenesis and liver regeneration after partial hepatectomy tonormal liver differentiation. Theses workers found changes in enzymeactivities during carcinogenesis that were similar to a step-wisereversal of differentiation. According to these workers, their resultssuggest that an underlying retrodifferentiation process is common toboth the process of hepatocarcinogenesis and liver regeneration.

[0031] More recently, Chastre et al (FEBS Letters [1985] vol 188, number2, pp 2810-2811] reported on the retrodifferentiation of the humancolonic cancerous subclone HT29-18.

[0032] Even more recently, Kobayashi et al (Leukaemia Research [1994]vol 18, no. 12, pp 929-933) have reported on the establishment of aretrodifferentiated cell line (RD-1) from a single ratmyelomonocyticleukemia cell which differentiated into a macrophage-likecell by treatment with lipopolysaccharide (LPS).

[0033] According to the current understanding, as borne out by theteachings found on page 911 of Molecular Biology of the Cell (pub.Garland Publishers Inc. 1983) and more recently Levitt and Mertelsman(ibid), a stem cell, such as a PSC, has the following fourcharacteristics:

[0034] i. it is an undifferentiated cell—i.e. it is not terminallydifferentiated;

[0035] ii. it has the ability to divide without limit;

[0036] iii. it has the ability to give rise to differentiated progeny,such as the differentiated cells mentioned earlier; and

[0037] iv. when it divides each daughter has a choice: it can eitherremain as PSC like its parent or it can embark on a course leadingirreversibly to terminal differentiation.

[0038] Note should be made of the last qualification, namely thataccording to the general teachings in the art once an undifferentiatedcell has differentiated to a more committed cell it can not thenretrodifferentiate. This understanding was even supported by theteachings of Uriel (ibid), Fukunda (ibid), Hass et al (ibid), Curtin andSnell (ibid), Chastre et al (ibid), and Kobayashi et al (ibid) as theseworkers retrodifferentiated certain types of differentiated cells butwherein those cells remained committed to the same lineage and they didnot retrodifferentiate into undifferentiated cells.

[0039] Therefore, according to the state of the art before the presentinvention, it was believed that it was not possible to formundifferentiated cells, such as stem cells, from more committed cells.However, the present invention shows that this belief is inaccurate andthat it is possible to form undifferentiated cells from more committedcells.

[0040] Thus, according to a first aspect of the present invention thereis provided a method of preparing an undifferentiated cell, the methodcomprising contacting a more committed cell with an agent that causesthe more committed cell to retrodifferentiate into an undifferentiatedcell.

[0041] According to a second aspect of the present invention there isprovided a provided a method comprising contacting a more committed cellwith an agent that causes the more committed cell to retrodifferentiateinto an undifferentiated cell; and then committing the undifferentiatedcell to a recommitted cell.

[0042] The term “recommitted cell” means a cell derived from theundifferentiated cell—i.e. a new more committed cell.

[0043] According to a third aspect of the present invention there isprovided an undifferentiated cell produced according to the method ofthe present invention.

[0044] According to a fourth aspect of the present invention there isprovided an undifferentiated cell produced according to the method ofthe present invention as or in the preparation of a medicament.

[0045] According to a fifth aspect of the present invention there isprovided the use of an undifferentiated cell produced according to themethod of of the present invention in the manufacture of a medicamentfor the treatment of an immunological disorder or disease.

[0046] According to a sixth aspect of the present invention there isprovided a recommitted cell produced according to the method of thepresent invention.

[0047] According to a seventh aspect of the present invention there isprovided a recommitted cell produced according to the method of thepresent invention as or in the preparation of a medicament.

[0048] According to an eighth aspect of the present invention there isprovided the use of a recommitted cell produced according to the methodof of the present invention in the manufacture of a medicament for thetreatment of an immunological disorder or disease.

[0049] According to a ninth aspect of the present invention there isprovided a more committed cell having attached thereto an agent that cancause the more committed cell to retrodifferentiate into anundifferentiated cell.

[0050] According to a tenth aspect of the present invention there isprovided a CD19⁺ and CD3⁺ cell.

[0051] Thus, in its broadest sense, the present invention is based onthe highly surprising finding that it is possible to form anundifferentiated cell from a more committed cell.

[0052] The present invention is highly advantageous as it is nowpossible to prepare undifferentiated cells from more committed cells andthen use those undifferentiated cells as, or to prepare, medicamentseither in vitro or in vivo or combinations thereof for the treatments ofdisorders.

[0053] The present invention is also advantageous as it is possible tocommit the undifferentiated cell prepared by retrodifferentiation to arecommitted cell, such as a new differentiated cell, with a view tocorrecting or removing the original more committed cell or forcorrecting or removing a product thereof.

[0054] Preferably, the more committed cell is capable ofretrodifferentiating into an MHC Class I⁺ and/or an MHC Class II⁺undifferentiated cell.

[0055] Preferably, the more committed cell is capable ofretrodifferentiating into an undifferentiated cell comprising a stemcell antigen.

[0056] Preferably, the more committed cell is capable ofretrodifferentiating into a CD34⁺ undifferentiated cell.

[0057] Preferably, the more committed cell is capable ofretrodifferentiating into a lymphohaematopoietic progenitor cell.

[0058] Preferably, the more committed cell is capable ofretrodifferentiating into a pluripotent stem cell.

[0059] The undifferentiated cell may comprise any components that areconcerned with antigen presentation, capture or recognition. Preferably,the undifferentiated cell is an MHC Class I⁺ and/or an MHC Class II⁺cell.

[0060] Preferably, the undifferentiated cell comprises a stem cellantigen.

[0061] Preferably, the undifferentiated cell is a CD34⁺ undifferentiatedcell.

[0062] Preferably, the undifferentiated cell is a lymphohaematopoieticprogenitor cell.

[0063] Preferably, the undifferentiated cell is a pluripotent stem cell.

[0064] The more committed cell may comprise any components that areconcerned with antigen presentation, capture or recognition. Preferably,the more committed cell is an MHC Class I⁺ and/or an MHC Class II⁺ cell.

[0065] Preferably, the agent acts extracelluarly of the more committedcell.

[0066] Preferably, the more committed cell comprises a receptor that isoperably engageable by the agent and wherein the agent operably engagesthe receptor.

[0067] Preferably, the receptor is a cell surface receptor.

[0068] Preferably, the receptor comprises an α-component and/or aβ-component.

[0069] Preferably, the receptor comprises a β-chain having homologousregions.

[0070] Preferably, the receptor comprises at least the homologousregions of the β-chain of HLA-DR.

[0071] Preferably, the receptor comprises an α-chain having homologousregions.

[0072] Preferably, the receptor comprises at least the homologousregions of the α-chain of HLA-DR.

[0073] Preferably, the agent is an antibody to the receptor.

[0074] Preferably, the agent is a monoclonal antibody to the receptor.

[0075] Preferably, the agent is an antibody, preferably a monoclonalantibody, to the homologous regions of the β-chain of HLA-DR.

[0076] Preferably, the agent is an antibody, preferably a monoclonalantibody, to the homologous regions of the α-chain of HLA-DR.

[0077] Preferably, the agent is used in conjunction with a biologicalresponse modifier.

[0078] Preferably, the biological response modifier is an alkylatingagent.

[0079] Preferably, the alkylating agent is or comprisescyclophosphoamide.

[0080] In one preferred embodiment, the more committed cell is adifferentiated cell.

[0081] Preferably, the more committed cell is any one of a B cell or a Tcell.

[0082] In an alternative preferred embodiment, the more committed cellis a more mature undifferentiated cell.

[0083] In one preferred embodiment, when the undifferentiated cell iscommitted to a recommitted cell the recommitted cell is of the samelineage as the more committed cell prior to retrodifferentiation.

[0084] In another preferred embodiment, when the undifferentiated cellis committed to a recommitted cell the recommitted cell is of adifferent lineage as the more committed cell prior toretrodifferentiation.

[0085] Preferably, the recommitted cell is any one of a B cell, a T cellor a granulocyte.

[0086] Preferably, the method is an in vitro method.

[0087] Preferably, the agent modulates MHC gene expression, preferablywherein the agent modulates MHC Class I⁺ and/or MHC Class II⁺expression.

[0088] The agent operably engages the more committed cell in order toretrodifferentiate that cell into an undifferentiated cell. In thisregard, the agent for the retrodifferentiation of the more committedcell into the undifferentiated cell may act in direct engagement or inindirect engagement with the more committed cell.

[0089] An example of direct engagement is when the more committed cellhas at least one cell surface receptor on its cell surface, such as aβ-chain having homologous regions (regions that are commonly foundhaving the same or a similar sequence) such as those that may be foundon B cells, and wherein the agent directly engages the cell surfacereceptor. Another example, is when the more committed cell has a cellsurface receptor on its cell surface such as an α-chain havinghomologous regions such as those that may be found on T cells, andwherein the agent directly engages the cell surface receptor.

[0090] An example of indirect engagement is when the more committed cellhas at least two cell surface receptors on its cell surface andengagement of the agent with one of the receptors affects the otherreceptor which then induces retrodifferentiation of the more committedcell.

[0091] The agent for the retrodifferentiation of the more committed cellinto an undifferentiated cell may be a chemical compound or composition.Preferably, however, the agent is capable of engaging a cell surfacereceptor on the surface of the more committed cell. For example,preferred agents include any one or more of cyclic adenosinemonophosphate (cAMP), a CD4 molecule, a CD8 molecule, a part or all of aT-cell receptor, a ligand (fixed or free), a peptide, a T-cell receptor(TCR), an antibody, a cross-reactive antibody, a monoclonal antibody, ora polyclonal antibody.

[0092] If the agent is an antibody, a cross-reactive antibody, amonoclonal antibody, or a polyclonal antibody, then preferably the agentis any one or more of an antibody, a cross-reactive antibody, amonoclonal antibody, or a polyclonal antibody to any one or more of: theβ chain of a MHC class II antigen, the β chain of a MHC HLA-DR antigen,the α chain of a MHC class I or class II antigen, the α chain of HLA-DRantigen, the α and the β chain of MHC class II antigen or of a MHC classI antigen. An example of a suitable antibody is CR3/43 (supplied byDako).

[0093] The more committed cell is any cell derived or derivable from anundifferentiated cell.

[0094] Thus, in one preferred embodiment, the more committed cell isalso an undifferentiated cell. By way of example therefore theundifferentiated cell can be a lymphoid stem cell or a myeloid stemcell, and the undifferentiated cell is a pluripotent stem cell.

[0095] In another preferred embodiment, the more committed cell is adifferentiated cell, such as a CFC-T cell, a CFC-B cell, a CFC-Eosincell, a CFC-Bas cell, a CFC-Bas cell, a CFC-GM cell, a CFC-MEG cell, aBFC-E cell, a CFC-E cell, a T cell, a B cell, an eosinophil, a basophil,a neutrophil, a monocyte, a megakaryocyte or an erythrocyte; and theundifferentiated cell is a myeloid stem cell, a lymphoid stem cell or apluripotent stem cell.

[0096] If the more committed cell is a differentiated cell thenpreferably the differentiated cell is a B lymphocyte (activated ornon-activated), a T lymphocyte (activated or nonactivated), a cell fromthe macrophage monocyte lineage, a nucleated cell capable of expressingclass I or class II antigens, a cell that can be induced to expressclass I or class II antigens or an enucleated cell (i.e. a cell thatdoes not contain a nucleus—such as a red blood cell).

[0097] In alternative preferred embodiments, the differentiated cell isselected from any one of a group of cells comprising large granularlymphocytes, null lymphocytes and natural killer cells, each expressingthe CD56 and/or CD16 cell surface receptors.

[0098] The differentiated cell may even be formed by the nucleation ofan enucleated cell.

[0099] The agent may act intracellularly within the more committed cell.However, preferably, the agent acts extracelluarly of the more committedcell.

[0100] In a preferred embodiment, agent operably engages a receptorpresent on the surface of the more committed cell—which receptor may beexpressed by the more committed cell, such as a receptor that is capablefo being expressed by the more committed cell.

[0101] Preferably, the receptor is a Class I or a Class II antigen ofthe major histocompatibility complex (MHC). In preferred embodiments thecell surface receptor is any one of: an HLA-DR receptor, a DM receptor,a DP receptor, a DQ receptor, an HLA-A receptor, an HLA-B receptor, anHLA-C receptor, an HLA-E receptor, an HLA-F receptor, or an HLA-Greceptor.

[0102] In more preferred embodiments the cell surface receptor is anHLA-DR receptor.

[0103] Preferably the contacting step comprises the agent engaging withany one or more of the following: homologous regions of the α-chain ofclass I antigens, homologous regions of the α-chain of class IIantigens, a CD4 cell surface receptor, a CD8 cell surface receptor,homologous regions of the β-chain of class II antigens in the presenceof lymphocytes, homologous regions of the α-chain of class I antigens inthe presence of lymphocytes, or homologous regions of the α-chain ofclass II antigens in the presence of lymphocytes.

[0104] Preferably the contacting step occurs in the presence of thebiological response modifier.

[0105] Preferably the biological response modifier is any one or more ofa modulator, such as an immunomodulator, a growth factor, a cytokine, acell surface receptor, a hormone, a nucleic acid, a nucleotide sequence,an antigen or a peptide.

[0106] In a preferred embodiment of the present invention theundifferentiated cell is then committed into a recommitted cell, such asa differentiated cell.

[0107] The recommitted cell may be of the same lineage to the morecommitted cell from which the undifferentiated cell was derived.

[0108] Alternatively, the recommitted cell may be of a different lineageto the more committed cell from which the undifferentiated cell wasderived.

[0109] In addition, the present invention also encompasses the method ofthe present invention for preparing an undifferentiated cell, whereinthe method includes committing the undifferentiated cell into arecommitted cell and then fusing the recommitted cell to a myeloma. Thisallows the expression in vitro of large amounts of the desired product,such as an antibody or an antigen or a hormone etc.

[0110] Other aspects of the present invention include:

[0111] The use of any one of the agents of the present invention forpreparing an undifferentiated cell from a more committed cell.

[0112] The use of an undifferentiated cell produced according to themethod of the present invention for producing any one of a monoclonal ora polyclonal or a specific antibody from a B-lymphocyte or aT-lymphocyte; a cell from the macrophage monocyte lineage; a nucleatedcell capable of expressing class I or class II antigens; a cell capableof being induced to express class I or class II antigens; an enucleatedcell; a fragmented cell; or an apoptic cell.

[0113] The use of an undifferentiated cell produced according to themethod of the present invention for producing effector T-lymphocytesfrom B-lymphocytes and/or vice versa.

[0114] The use of an undifferentiated cell produced according to themethod of the present invention for producing any one or more of: amedicament, such as a medicament comprising or made from a B-lymphocyte,a T-lymphocyte, a cell from the macrophage monocyte lineage, a nucleatedcell capable of expressing a class I or a class II antigen, a cellcapable of being induced to express a class I or a class II antigen, oran enucleated cell.

[0115] The present invention also encompasses processes utilising theafore-mentioned uses and products or compositions prepared from suchprocesses.

[0116] The present invention also encompasses a medicament comprising anundifferentiated cell according to the present invention or a productobtained therefrom admixed with a suitable diluent, carrier orexcipient.

[0117] In one preferred embodiment the medicament comprises an antibodyor antigen obtained from an undifferentiated cell according to thepresent invention admixed with a suitable diluent, carrier or excipient.

[0118] Preferably the medicament is for the treatment of any one of:cancer, autoimmune diseases, blood disorders, cellular or tissueregeneration, organ regeneration, the treatment of organ or tissuetransplants, or congenital metabolic disorders.

[0119] In a preferred embodiment the present invention relates to aprocess of introducing a gene into the genome of an undifferentiatedcell, wherein the process comprises introducing the gene into a morecommitted cell, and then preparing an undifferentiated cell by themethod according to the present invention, whereby the gene is presentin the undifferentiated cell.

[0120] In a more preferred embodiment the present invention relates to aprocess of introducing a gene into the genome of an undifferentiatedcell, wherein the process comprises inserting the gene into the genomeof a more committed cell, and then preparing an undifferentiated cell bythe method according to the present invention, whereby the gene ispresent in the undifferentiated cell.

[0121] In an even more preferred embodiment the present inventionrelates to a process of introducing the genome of a gene into anundifferentiated cell, wherein the process comprises inserting the geneinto the genome of a more committed cell, and then preparing anundifferentiated cell by the method according to the present invention,whereby the gene is present in the genome of the undifferentiated cell.

[0122] The present invention encompasses an undifferentiated cellprepared by any one of these processes of the present invention.

[0123] As already mentioned, the present invention also encompasses amedicament comprising an undifferentiated cell prepared by any one ofthese processes admixed with a suitable diluent, carrier or excipient.With such a medicament the undifferentiated cell could be used toproduce a beneficial more committed cell, such as one having a correctgenomic structure, in order to alleviate any symptoms or conditionsbrought on by or associated with a more committed cell having anincorrect genomic structure.

[0124] Thus, the present invention also provides a process of removingan acquired mutation from a more committed cell wherein the methodcomprises forming an undifferentiated cell by the method according tothe present invention, committing the undifferentiated cell into arecommitted cell, whereby arrangement or rearrangement of the genomeand/or nucleus of the cell causes the mutation to be removed.

[0125] Preferably the gene is inserted into the immunoglobulin region orTCR region of the genome.

[0126] Alternatively, the undifferentiated cell could be used to producea more committed cell that produces an entity that cures any symptoms orconditions brought on by or associated with a more committed cell havingan incorrect genomic structure. For example, the present invention maybe used to prepare antibodies or T cell receptors to an antigen that isexpressed by the more committed cell which has retrodifferentiated intothe undifferentiated cell. In this regard, the antigen may be afetospecific antigen or a cross-reactive fetospecific antigen.

[0127] The present invention also includes a process of controlling thelevels of undifferentiated cells and more committed cells. For example,the present invention includes a method comprising forming anundifferentiated cell by the method according to the present inventionand then activating an apoptosis gene to affect the undifferentiatedcell, such as bring about the death thereof.

[0128] In one preferred embodiment of the present invention, the morecommitted cell is not a cancer cell. In another preferred embodiment ofthe present invention, the agent is neither carcinogenic nor capable ofpromoting cancer growth.

[0129] The present invention also covers a method of treating a patientsuffering from a disease or a disorder resulting from a defective cellor an unwanted cell, the method comprising preparing an undifferentiatedcell by contacting a more committed cell with an agent that causes themore committed cell to retrodifferentiate into the undifferentiatedcell, and then optionally committing the undifferentiated cell into arecommitted cell; wherein the undifferentiated cell, or the recommittedcell, affects the defective cell or the unwanted cell to alleviate thesymptoms of the disease or disorder or to cure the patient of thedisease or condition.

[0130] In summation, the present invention relates to the preparation ofan undifferentiated cell from a more committed cell.

[0131] The present invention will now be described by way of example, inwhich reference shall be made to the following Figures:

[0132]FIG. 1 which is a microscope picture of cells before the method ofthe present invention;

[0133]FIG. 2 which is a microscope picture of cells prepared by themethod of the present invention;

[0134]FIG. 3 which is a microscope picture of cells prepared by themethod of the present invention but at a lower magnification;

[0135]FIG. 4 which is a microscope picture of cells before the method ofthe present invention;

[0136]FIG. 5 which is a microscope picture of cells prepared by themethod of the present invention; and

[0137]FIG. 6 which is a microscope picture of cells prepared by themethod of the present invention.

A. MATERIALS AND METHODS

[0138] Patients

[0139] Blood samples were obtained in lavender top tubes containing EDTAfrom patients with B-cell chronic lymphocytic leukaemia's, patients withantibody deficiency (including IgA deficiency and X-linked infantilehypogammaglobulinaemias), patients with HIV infections and AIDSsyndrome, a patient with CMV infection, a patient with Hodgkin'slymphomas, a patient with acute T-cell leukaemia, a 6-days old baby withHodgkin's lymphomas, a patient with acute T-cell leukaemia, a 6-days oldbaby with blastcytosis, various patients with various infections andclinical conditions, cord blood, bone marrow's, and enrichedB-lymphocyte preparations of healthy blood donors.

[0140] Clinical and Experimental Conditions

[0141] The clinical and experimental treatment conditions of patients,including various types of treatment applied to their blood samples, aredescribed in Table 1. Differential white blood cell (WBC) counts wereobtained using a Coulter Counter and these are included in the sameTable.

[0142] Treatment of Blood

[0143] Blood samples, once obtained, were treated with pure monoclonalantibody to the homologous region of the β-chain of the HLA-DR antigen(DAKO) and left to mix on a head to head roller at room temperature fora maximum of 24 hours. Some samples were mixed first on a head to headroller for 15 minutes after which they were left to incubate in anincubator at 22° C. The concentration of monoclonal antibody added toblood samples varied from 10-50 μl/ml of blood.

[0144] In addition, other treatments treatments were applied at the sameconcentrations and these included addition of a monoclonal antibody tothe homologous of the α-chain of the HLA-DR antigen, a monoclonalantibody to the homologous region of class I antigens, a monoclonalantibody to CD4, a monoclonal antibody to CD8, and a PE conjugatedmonoclonal antibody to the homologous region of the β-chain of theHLA-DR antigen.

[0145] Other treatments included the simultaneous addition of monoclonalantibodies to the homologous regions of the α and β-chains of the HLA-DRantigen to blood samples.

[0146] Furthermore, alkylating agents such as cyclophosphoamide wereadded to blood samples in combination with pure monoclonal antibody tothe homologous region of the β-chain of the HLA-DR antigen.

[0147] Following these treatments blood samples were stained with panelsof labelled monoclonal antibodies as instructed by the manufacturer'sinstructions and then analyzed using flow cytometry.

[0148] Incubation periods with monoclonal antibodies ranged from 2 hour,4 hour, 6 hour, 12 hour to 24 hour intervals.

[0149] Labelled Antibodies

[0150] The following monoclonal antibodies were used to detect thefollowing markers on cells by flow cytometry: CD19 and CD3, CD4 and CD8,DR and CD3, CD56 & 16 and CD3, CD45 and CD14, CD8 and CD3, CD8 and CD28,simultest control (IgG1 FITC+IgG2a PE), CD34 and CD2, CD7 and CD13 & 33,CD10 and CD25, CD5 and CD10, CD5 and CD21, CD7 and CD5, CD13 and CD20,CD23 and CD57 and CD25 and CD45 RA (Becton & dickenson and DAKO).

[0151] Each patient's blood sample, both treated and untreated, wasanalyzed using the majority of the above panel in order to account forthe immunophenotypic changes that accompanied different types oftreatments and these were carried out separately on different aliquotsof the same blood sample. Untreated samples and other control treatmentswere stained and analyzed simultaneously.

[0152] Flow Cytometry

[0153] Whole blood sample was stained and lysed according to themanufacturer instructions. Flow cytomery analysis was performed on aFACScan@ with either simultest or PAINT A GATE software (BDIS) whichincluded negative controls back tracking. 10,000 to 20,000 events wereacquired and stored in list mode files.

[0154] Morphology

[0155] Morphology was analyzed using microscopy and Wright's stain.

B. RESULTS

[0156] CD19 and CD3 Panel

[0157] Treatment of blood samples with monoclonal antibody to thehomologous region of the β-chain of the HLA-DR antigen always decreasedthe relative number of CD19⁺ cells. This marker is a pan B-cell antigen(see Table). This antigen is present on all human B lymphocytes at allstages of maturation but is lost on terminally differentiated plasmacells. Hence, this is an indication that B cells wereretrodifferentiating into undifferentiated cells.

[0158] The same treatment caused the relative number of CD3⁺ cells toincrease dramatically especially in blood of patients with B-CLL, whichwas always accompanied by an increase in the relative number inCD3⁻CD19⁻ cells. CD3 is present on all mature T-lymphocytes and on65%-85% of thymocytes. This marker is always found in association withα-/β- or gamma/delta T-cell receptors (TCR) and together these complexesare important in transducing signals to the cell interior. Hence, thisis an indication that B cells were retrodifferentiating intoundifferentiated cells and then being committed to new differentiatedcells, namely T cells.

[0159] A novel clone of cells appeared in treated blood of B-CLLpatients co-expressing the CD19 and CD3 markers—i.e. CD19⁺ and CD3⁺cells (see Charts 1, patient 2, 3 & 4 at 2 hr, 6 hr & 24 hr of startingtreatment). Other patients with different conditions showed an increasein the relative number of these clones of cells. These cells wereexceptionally large and heavily granulated and extremely high levels ofCD19 were expressed on their cell membrane. The CD3 marker seems to beexpressed on these cells at similar levels to those expressed on normalmature lymphocytes.

[0160] In Table 2, patient numbers 2, 3 and 4 are actually numbersrepresenting the same patient and their delineation was merely to showthe effect of treatment on blood with time (See Table 1 for experimentaland clinical condition of this patient).

[0161] The CD19⁺CD3⁺ clones in treated samples seem to decrease withtime, reaching original levels to those determined in untreated sampleat 2 hrs, 6 hrs and 24 hrs time.

[0162] Another type of cell of the same size and granulity was detectedin treated samples and these cells had high levels of CD19 expressed ontheir surface but were negative for the CD3 marker and rich in FCreceptors. However, the relative number of these cells appeared todecrease in time. Of interest, at 24 hours treatment of blood sample (2,3 and 4) there was a decrease in the relative number of CD19⁻CD3⁻ cellsin a group of cells that were initially observed to increase after 2 and6 hr's treatment of blood samples. However, Coulter counts of WBCpopulations were reduced on treatment of blood with monoclonal antibodyto the homologous region of the β-chain of the HLA-DR antigen. Thisfinding suggests that this type of treatment gives rise to atypicalcells that cannot be detected by Coulter (Table 1) but can be accountedfor when measured by flow cytometry which counts cells on the basis ofsurface markers, size and granulity. Furthermore, these atypical cellswere accounted for by analysing morphology using Wright's stain under amicroscope. Flow cytometric charts of these phenomena are represented inCharts (1, 2, 3 & 4) and the immunophenotypic changes obtained ontreatment of blood samples seems to suggest that CD19⁺ and CD3⁺lymphocytes are an interconnected group of cells but remain distinct onthe basis of CD19 and CD3 relative expression compared to stem cells.

[0163] In Table 2, patient numbers 5 and 6 represent the same patientbut analysis of treated and untreated blood samples were monitored withtime and at the same time (see Table 1).

[0164] Patients blood with no B-cell malignancy showed similar trends ofimmunophenotypic changes when compared to blood of B-CLL patients butthe changes were not to the same extent. However, the relative andabsolute number of B-lymphocytes and MHC class II positive cells in theblood of these patients are extremely low compared to those found in theblood of B-CLL patients.

[0165] Two brothers both with X-linked infantile hypogammaglobulinemiawho were B cell deficient showed different immunophenotypic changes inthe relative number of CD3⁺ cells on treatment of their blood. Theyounger brother who was 2 months old and not ill, on treatment of hisblood, showed a slight increase in the relative number of CD3⁺ cellswhich was accompanied by a decrease in the relative number of CD3⁻CD19⁻cells. On the other hand, the other brother who was 2 years old and wasextremely sick and with a relatively high number of activated T cellsexpressing the DR antigens showed a decrease in the number of CD3⁺ cellson treatment of his blood. No other markers were used to measure otherimmunophentypic changes that might have occurred because the bloodsamples obtained from these two patients were extremely small (Table 2,ID 43/BD and 04/BD).

[0166] Patient 91 in Table 2 shows a decrease in the relative number ofCD3⁺ cells following treatment of blood which was accompanied by anincrease in the relative number of CD3⁻CD19⁻ cells. However, on analysisof other surface markers such as CD4 and CD8 (see Table 3) the patientwas observed to have a high relative number of CD4⁺CD8⁻ cells in hisblood and this was noted prior to treatment of blood samples withmonoclonal antibody to the β-chain of the DR antigen and these doublepositive cells decreased appreciably following treatment of blood.Furthermore, when further markers were analyzed the relative number ofCD3⁺ cells were seen to have elevated (See Table 4).

[0167] An enriched preparation of B-lymphocytes obtained from healthyblood donors when treated with monoclonal antibody to the β-chain of DRantigens showed a dramatic increase in the relative number of CD3⁺ cellswhich were always accompanied by a decrease in the relative number ofCD19⁺ cells and by an increase in the relative number of CD19⁻CD3⁻cells. Further analysis using markers such as CD4 and CD8 show aconcomitant increase in the relative number of these markers. However,an enriched preparation of T lymphocytes of the same blood donors whentreated with the same monoclonal antibody did not show the same changes.

[0168] CD4 and CD8 Panel

[0169] The CD4 antigen is the receptor for the human immunodificiencyvirus. The CD4 molecule binds MHC class II antigen in the B2 domain, aregion which is similar to the CD8 binding sites on class I antigens.Binding of CD4 to class II antigen enhances T cell responsiveness toantigens and so does the binding of CD8 to class I antigens. The CD8antigens are present on the human supressor/cytotoxic T-lymphocytessubset as well as on a subset of natural killer (NK) lymphocytes and amajority of normal thymocytes. The CD4 and CD8 antigens are coexpressedon thymocytes and these cells lose either markers as they mature intoT-lymphocytes.

[0170] On analysis of the CD4 and CD8 markers—see below—and from amajority of blood samples presented in Table 2, a pattern of stainingemerges which supports the presence of a retrodifferentiation process ofB-lymphocytes into undifferentiated cells and the subsequentdifferentiation into T-lymphocytes. CD4⁺CD8⁺ cells, which are doublepositive cells, always appeared following treatment of blood sampleswith monoclonal antibody to the homologous region of the β-chain andthese types of cells were markedly increased in the blood of treatedsamples of patients with B-CLL and which were absent altogether inuntreated samples (See Table 3 and Charts 1, 2 3 & 4). In the samespecimens the relative number of single positive cells such as CD8⁺ andCD4⁺ cells was also noted to increase simultaneously. Furthermore, adecrease in the relative number of CD4⁻CD8⁻ cells which, at least in thecase of B-CLL correspond to B cells was noted to fall dramatically intreated samples when compared to untreated specimens which remained atthe same level when measured with time. However, measurement of therelative number of CD4⁺CD8⁺ cells with time in treated samples showedthat there was a concomitant increase in the number of single positivecells with a decrease in the relative number of double positive cells.This type of immunophenotypic change is characteristic of thymicdevelopment of progenitor cells of the T-lymphocyte lineage in thethymus (Patient number 2,3 and 4). The CD4 antigen is present on thehelper/inducer T-lymphocyte subsets (CD4⁻CD3 ⁺) and a majority of normalthymocytes. However, this antigen is present in low density on the cellsurface of monocytes and in the cytoplasm of monocytes and macrophages(CD3⁻CD4⁺).

[0171] The relative number of CD4⁺ low cells was affected differently indifferent blood samples following treatment. The relative number of thistype of cells seems unaffected in blood samples of patients with B-CLLfollowing treatment when compared to untreated samples. Such low levelsof CD4 expression is found on monocytes and very early thymocytes.

[0172] Patient HIV⁺25 on treatment showed a substantial increase in thenumber of double positive cells expressing CD4 and CD8 simultaneously.On the other hand, patient 91 on treatment showed a decrease in thissubtype of cells and the observation of such phenomenon is timedependent. The relative number of CD8⁺ cells was observed to increase inuntreated blood samples of patients with B-CLL when measured with timewhereas the relative number of CD4⁺ and CD4⁺ low cells was observed todecrease at the same times (Table 3 patient 2,3 and 4).

[0173] DR and CD3 Panel

[0174] The DR markers are present on monocytes, dendritic cells, B-cellsand activated T-lymphocytes.

[0175] Treated and untreated samples analysed with this panel showedsimilar immunophenotypic changes to those obtained when blood sampleswere analysed with the CD19 and CD3 markers (see Table 2) and theseantigens as mentioned earlier are pan B and T-cell markers respectively.

[0176] Treatment of blood with monoclonal antibodies seems to affect therelative number of DR⁺ B-lymphocytes so that the level of DR+ cellsdecrease. In contrast, the relative number of CD3⁺ (T-cells) cellsincrease significantly (see Table 4 and Chart).

[0177] Furthermore, the relative number of activated T cells increasedin the majority of treated blood samples of patients with B-CLL andthese types of cells were affected variably in treated samples ofpatients with other conditions. Furthermore, the relative number of DRhigh positive cells appeared in significant numbers in treated samplesof patients with B-CLL and a 6 day old baby with increased DR⁺CD34⁺blasts in his blood. However, it should be noted that the blasts whichwere present in this patient's blood were negative for T and B-cellmarkers before and after treatment but became more positive for myeloidlineage antigens following treatment. The relative number of CD3⁻DR⁻cells increased in the majority of treated blood samples and wasproportional to increases in the relative number of CD3⁺ cells (T-cells)and was inversely proportional to decreases in the relative number ofDR+ cells (B-cells).

[0178] CD56&16 and CD3 Panel

[0179] The CD56&CD16 markers are found on a heterogeneous group ofcells, a subset of lymphocytes known generally as large granularlymphocytes and natural killer (NK) lymphocytes. The CD16 antigen isexpressed on virtually all resting NK lymphocytes and is weaklyexpressed on some CD3⁺ T lymphocytes from certain individuals. Thisantigen is found on granulocytes in lower amount and is associated withlymphocytes containing large azurophilic granules. The CD16 antigen isthe IgG FC receptor III.

[0180] A variable number of CD16⁺ lymphocytes coexpress either the CD57antigen or low-density CD8 antigen or both. In most individuals, thereis virtually no overlap with other T-lymphocyte antigens such as theCD5, CD4, or CD3 antigens. The CD56 antigen is present on essentiallyall resting and activated CD16⁺ NK lymphocytes and these subsets ofcells carry out non-major histocompatibility complex restrictedcytotoxicity.

[0181] Immunophenotyping of treated and untreated blood samples of B-CLLand some other patients with other conditions showed an increase in therelative number of cells coexpressing the CD56& CD16 antigens which wereheavily granulated and of medium size (see Table 5 and Charts 1, 2, 3 &4). These observations were also accompanied by a marked increase in therelative number of cells expressing the CD3 antigen only (without theexpression of CD56 and CD16 markers) and cells coexpressing theCD56&CD16 and CD3 markers together.

[0182] In Table 5, patient numbers 2, 3, and 4 represent the same bloodsample but being analysed at 2 hours, 6 hours and 24 hours respectively(before and after treatment). This sample shows that treatment of bloodwith monoclonal antibody to the homologous region of the β-chain of DRantigen seems to cause spontaneous production of CD56⁺ and CD16⁺ cells,CD3⁺ cells and CD56 ⁺ and CD16⁺CD3⁺ cells and these observations werealways accompanied by the disappearance of B-cell markers (CD19, DR,CD56, CD16⁻CD3⁻).

[0183] Onward analysis of this blood sample before and after treatmentshowed the levels of CD56⁺ and CD16⁺ cells to decrease with time and thelevel of CD3⁺ cells to increase with time.

[0184] Blood samples of patient 7 with B-CLL, did not show any changesin the number of cells expressing the CD56, CD16 and CD3 antigens whencompared to immnunophenotypic changes observed in treated and untreatedsamples and this is because the amount of monoclonal antibody added wasextremely low relative to the number of B lymphocytes. However,treatment of this patient's blood sample on a separate occasion with anappropriate amount of monoclonal antibody showed significant increasesin the relative number of CD3⁺, CD56⁺ & CD16⁺ and CD56⁺ and CD16⁺CD3⁺cells.

[0185] Blood samples of other patients with other conditions showedvariable changes in the level of these cells and this seems to bedependent on the number of B-lymphocytes present in blood beforetreatment, duration of treatment and probably the clinical condition ofpatients.

[0186] CD45 and CD14 Panel

[0187] The CD45 antigen is present on all human leukocytes, includinglymphocytes, monocytes, polymorphonuclear cells, eosinophils, andbasophils in peripheral blood, thymus, spleen, and tonsil, and leukocyteprogenitors in bone marrow.

[0188] The CD14 is present on 70% to 93% of normal peripheral bloodmonocytes, 77% to 90% of pleural or peritoneal fluid phagocytes. Thisantigen is weakly expressed on granulocytes and does not exist onunstimulated lymphocytes, mitogen-activated T lymphocytes, erythrocytes,or platelets.

[0189] The CD45 antigen represents a family of protein tyrosinephosphatases and this molecule interacts with external stimuli(antigens) and effects signal transduction via the Scr-family membersleading to the regulation of cell growth and differentiation.

[0190] Engagement of the β-chain of the DR antigens in treated bloodsamples especially those obtained from patients with B-CLL suggests thatsuch a treatment affects the level of CD45 antigens on B-lymphocytes.The overall immunophenotypic changes that took place on stimulation ofthe β-chain of the DR antigen seem to give rise to different types ofcells that can be segregated on the basis of the level of CD45 and CD14expression as well as morphology as determined by forward scatter andside scatter (size and granulity respectively) and these results arepresented in Table 6 and Charts (1, 2, 3, 4 & 5).

[0191] On treatment the relative number of CD45 low cells (when comparedto untreated samples) increased significantly and so did the relativenumber of cells co-expressing the CD45 and CD14 antigens. This type ofimmunophenotypic changes coincided with a decrease in the relativenumber of CD45 high cells (compared to untreated samples). However, thislatter population of cells can be further divided on the basis ofmorphology and the degree of CD45 expression. One type was extremelylarge and had extremely high levels of CD45 antigen when compared to therest of cells present in the charts (see charts 1, 2, 3 and 4). Onanalysis of this panel following treatment with time (see Table patient2,3 and 4 and charts) the relative number of CD45⁺ cells initially felldrastically with time to give rise to CD45 low cells. However, analysisof blood 24 hours later showed the opposite situation.

[0192] Samples 5 and 7 reveal opposite immunophenotypic changes to thoseobtained with other samples obtained from other B-CLL patients and thisis because the samples were analysed at a much earlier incubation timewith the monoclonal antibody. In fact the sequential analysis of bloodsamples after treatment seems to suggest that the immunophenotypicchanges undertaken by B lymphocytes is time dependent because itrepresents a stage of development and the immunophenotypic changesmeasured at time X is not going to be the same at time X plus (its notfixed once induced). However, these types of changes must be occurringin a more stringent manner in the body otherwise immunopathology wouldensue. The effect of treatment of blood samples from other patients withno B-cell malignancy show variable changes in immunophenotypes of cellsand this because B-lymphocytes are present in lower amount. However,treatment of enriched fractions of B-lymphocytes obtained from healthyblood donors show similar immunophenotypic changes to those obtainedwith B-CLL with high B lymphocyte counts.

[0193] CD8 and CD3 Panel

[0194] The CD8 antigenic determinant interacts with class I MHCmolecules, resulting in increased adhesion between the CD8 + Tlymphocytes and the target cells. This type of interaction enhances theactivation of resting lymphocytes. The CD8 antigen is coupled to aprotein tyrosine kinase (p56ick) and in turn the CD8/p56ick complex mayplay a role in T-lymphocyte activation.

[0195] Treatment of blood samples obtained from patients with B-CLL withmonoclonal antibody to the B chain causes a significant increase in therelative number of CD3CD8 and CD3 (highly likely to be CD4CD3) positivecells thus indicating more clearly that double positive cells generatedinitially are undergoing development into mature T-lymphocytes. This isa process that can be measured directly by CD19 and by DR and indirectlyby CD8⁻CD3⁻ antigens. Serial assessment of treated blood samples of thesame patient with time seems to agree with a process which is identicalto thymocyte development (Table 7, patient 2, 3 and 4 and Chart 1).

[0196] The relative number of CD8⁺ cells increased with time in treatedand untreated samples but to a higher extent in untreated samples. Onthe other hand, the relative number of CD8⁺CD3⁺ cells decreased withtime in untreated samples. However, the relative number of CD3⁺ cellsincreased in treated blood samples when measured with time and thesetypes of cells highly correspond to CD4⁺CD3⁺ single positive cells; amaturer form of thymocytes. In addition, since these samples were alsoimmunophenotyped with other panels (mentioned above in Tables 3, 4, 5and 6) the overall changes extremely incriminate B cells in thegeneration of T lymphocyte progenitors and progenies.

[0197] Blood samples from a patient with B-CLL (number 2, 3 and 4 Tables1, 2, 3, 4, 5, 6, 7) in separate aliquots were treated with nothing, PEconjugated monoclonal antibody to the homologous region of the β-chainof DR antigen and unconjugated form of the same monoclonal antibody. Oncomparison of PE conjugated treatment clearly indicates no change in therelative number of CD3 positive cells and associated markers such as CD4which have been observed in significant levels when the same bloodsample was treated with unconjugated form of the antibody. However, anincrease in the number of CD45 positive cells with no DR antigen beingexpressed on their surface was noted when measured with time (see Table8). A finding that was similar to that noted in untreated samples whenimmunophenotyped with time (Table 6). Furthermore, the relative numberof cells expressing CD45 low decreased in time, a phenomenon which wasalso noted in the untreated samples (when measured with time) of thesame patient (see chart 1A).

C. COMPARISON OF THE EFFECT OF OTHER MONOCLONAL ANTIBODIES WITHDIFFERENT SPECIFICITY ON T-LYMPHOPHOIESIS

[0198] CD19 and CD3 Panel

[0199] Treatment of blood samples with monoclonal antibody to thehomologous region of the α-chain of the DR antigen and the homologousregion of MHC Class I antigens decreased the number of CD3⁺ cells andincreased the number of CD19⁺ cells. Treatment of the same blood withmonoclonal antibody to the homologous region of the β-chain of the DRantigen decreased the number of CD19⁺ cells and increased the number ofCD3⁺ cells. Treatment with the latter monoclonal antibody withcyclophosphoamide revealed the same effect (Table 14 patient 5/6 withB-CLL at 2 hr treatment).

[0200] Onward analysis of CD19⁺ and CD3⁺ cells in the same samplesrevealed further increases in the relative number of CD3⁺ cells only inblood treated with monoclonal antibody to the homologous region of theβ-chain of DR antigen (Table 14 patient 5/6 at 24 hours followingtreatment). However, onward analysis (24 hours later patient 5/6 Table14) of blood samples treated with cyclophosphamide plus monoclonalantibody to the β-chain of DR antigen show reversal in the relativenumber of CD19⁺ and CD⁺ cells when compared to that observed at 2 hourincubation time under exactly the same condition.

[0201] In general, treatment of blood samples of the same patient withmonoclonal antibody to the homologous region of the α chain of the DRantigen or monoclonal antibody to the homologous of the α-chain of theclass I antigen shows an increase in the relative number of CD19⁺ cells(pan B marker) when compared to untreated sample. The relative number ofCD19⁻CD3⁻ cells decreased slightly in blood samples treated withmonoclonal antibody to the α-chain of DR antigen or treated withmonoclonal antibody to class I antigens (see Table 14 & Charts 2, 3 &4). Treatment of blood samples of patient 09 with monoclonal antibody toclass I antigens increased the relative number of CD3⁺ cells anddecreased slightly the relative number of CD19⁺ and CD19⁻CD3⁻ cells.However, treatment of an enriched preparation of B-lymphocytes obtainedfrom healthy blood donors with monoclonal antibody to the β-chain orα-chain of DR antigen showed similar immunophenotypic changes to thoseobtained with patient with B-CLL.

[0202] Treatment of HIV⁺ and IgA deficient patients with monoclonalantibody to the β-chain of the DR antigen increased the relative numberof CD3⁺ cells and decreased the relative number of CD19⁺ cells. However,treatment of the same blood sample with monoclonal antibody to thehomologous region of class I antigen did not produce the same effect.Treatment of blood samples obtained from patients (34/BD and 04/BD) withB-cell deficiency showed variable immunophenotypic changes when treatedwith monoclonal antibodies to the β-chain of the DR antigen, class Iantigens and CD4 antigen.

[0203] CD4 and CD8 Panel

[0204] Blood samples analysed using the CD19 and CD3 panel (Table 14)were also immunophenotyped with the CD4 and CD8 panel (Table 15). Bothpanels seem to agree and confirm each other. Incubation for 2 hours ofblood samples of patients with B-CLL (Table 15, patients 5/6 and 10,Charts 2, 3 & 4) with monoclonal antibody to the homologous region ofthe β-chain of the DR antigen or with this monoclonal antibody pluscyclophosphoamide increased the relative number of CD8⁺ and CD4⁺ cellsand cells coexpressing both markers. On the other hand, treatment of thesame samples with monoclonal antibodies to the homologous region of theα-chain of the DR antigen or the homologous region of the α-chain ofclass I antigen did not produce the same effects.

[0205] Comparison of immunophenotypic trends obtained at 2 hours and 24hours incubation periods with monoclonal antibody to the β-chain of theDR antigen plus cyclophosphoamide revealed reverse changes in therelative number of CD4 and CD8 positive cells (Table 15, patient 5/6with B-CLL at 2 hours and 24 hours) and such changes were in accordancewith those obtained when the same blood sample was analysed with theCD19 and CD3 panel (Table 14 the same patient). The later findingsindicate that the subsequent differentiation is reversible as theundifferentiated cells can differentiate into T-lymphocytes orB-lymphocytes.

[0206] DR and CD3 Panel

[0207] The immunophenotypic changes obtained with DR and CD3 (Table 16)panel confirm he findings obtained with CD19 and CD3 panel and CD4 andCD8 panel (Tables 14 & 15 & Charts 2, 3 & 4) which followed treatment ofthe same blood samples with monoclonal antibodies to the homologousregion of the beta- or alpha-side of the DR antigen or monoclonalantibody to class I antigens or monoclonal antibody to the β-chain ofthe DR antigen plus cyclophosphoamide at 2 hour analysis.

[0208] From the results, it would appear that the monoclonal antibody tothe homologous region of the β-chain of the DR antigen is extremelycapable of driving the production of CD3 positive cells from DR⁺ cells.

[0209] Furthermore, treatments such as those involving engagement of theα-chain of DR antigens or engagement of the β-side of the molecule inconjunction with cyclophosphoamide (prolonged incubation time) promotedincreases in the relative number of CD19⁺ cells or DR⁺ cells.

[0210] CD56&16 and CD3 Panel

[0211] Treatment of blood samples, especially of those of patients withB-CLL with high B-lymphocyte counts with monoclonal antibody to thehomologous region of the β-chain of the DR antigen increased therelative number of CD56&16 positive cells.

[0212] In these patients the relative number of CD3⁺ and CD56⁺ andCD16⁺CD3⁺ cells also increased following treatment of blood samples withmonoclonal antibody to the β-chain, confirming earlier observationsnoted with the same treatment when the same blood samples were analysedwith CD3 and CD19 and DR and CD3 panels.

[0213] CD45 and CD14 Panel

[0214] Blood samples treated with monoclonal antibodies to the β- oralpha-chains of the DR antigen or to the β-chain plus cyclophosphoamideor class I antigens were also analysed with the CD45 and CD14 panel(Table 18). The delineation of CD45 low, CD45 high and CD45 medium isarbitrary. Treatment of blood sample 5/6 (at 2 hours) with monoclonalantibodies to the β-chain of the DR antigen or with this monoclonalantibody plus cyclophosphoamide generated CD45⁺ low cells and increasedthe relative number of CD45⁺ medium cells. However, the former treatmentincreased the relative number of CD45⁺ high cells and the lattertreatment decreased the relative number of CD45⁺ medium cells and thesechanges appeared to be time dependent.

[0215] Blood samples of patient 5/6 and 10 (B-CLL) on treatment withmonoclonal antibody to class I antigens showed a decrease in therelative number of CD45⁺ medium cells and similar observations werenoted in blood samples 09 and HIV⁺ following the same treatment whencompared to untreated samples. Treatment of blood samples of HIV⁺ andIgA/D patients with monoclonal antibody to class I antigen increased therelative number of CD45⁺ low cells when compared to untreated samples orsamples treated with monoclonal antibody to the β-chain of the DRantigen. However, blood samples of these patients showed a decrease inthe relative number of CD45⁺ medium cells on treatment with monoclonalantibody to the homologous regions of the β-chain of the DR antigen.Medium CD45⁺ cells increased in blood samples of IgA/D patient followingmonoclonal antibody to class I antigen treatment. Cells that wereextremely large, heavily granular and expressing intense levels of CD45antigen were noted in treated blood samples with monoclonal antibody tothe homologous region of the β-chain of DR antigen of MHC class IIantigens (see Charts 1, 2, 3, 4 & 5).

[0216] CD8 and CD28 Panel

[0217] The CD28 antigen is present on approximately 60% to 80% ofperipheral blood T (CD3⁺) lymphocytes, 50% of CD8⁺ T lymphocytes and 5%of immature CD3-thymocytes. During thymocyte maturation, CD28 antigenexpression increases from low density on most CD4⁺CD8⁺ immaturethymocytes to a higher density on virtually all mature CD3⁺, CD4⁺ orCD8⁺ thymocytes. Cell activation further augments CD28 antigen density.Expression of the CD28 also d vides the CD8⁺ lymphocytes into twofunctional groups. CD8⁺CD28⁺ lymphocytes mediate alloantigen-specificcytotoxicity, that is major histocompatibility complex (MHC) classI-restricted. Suppression of cell proliferation is mediated by theCD8⁺CD28⁻ subset. The CD28 antigen is a cell adhesion molecule andfunctions as a ligand for the B7/BB-1 antigen which is present onactivated B lymphocytes.

[0218] Treatment of blood samples of patients (Table 19, patients 5/6and 8) with B-CLL with monoclonal antibody to the homologous region ofβ-chain of the DR antigen increased the relative number of CD8⁺, CD28⁺and CD8⁺CD28⁺ cells and all other types of treatments did not.

[0219] CD34 and CD2 Panel

[0220] The CD34 antigen is present on immature haematopoietic precursorcells and all haematopoietic colony-forming cells in bone marrow,including unipotent (CFU-GM, BFU-E) and pluripotent progenitors(CFU-GEMM, CFU-Mix and CFU-blast). The CD34 is also expressed on stromalcell precursors. Terminal deoxynucleotidyl transferase (TdT)⁺ B- andT-lymphoid precursors in normal bone are CD34⁺, The CD34 antigen ispresent on early myeloid cells that express the CD33 antigen but lackthe CD14 and CD15 antigens and on early erythroid cells that express theCD71 antigen and dimly express the CD45 antigen. The CD34 antigen isalso found on capillary endothelial cells and approximately 1% of humanthymocytes. Normal peripheral blood lymphocytes, monocytes, granulocytesand platelets do not express the CD34 antigen. CD34 antigen density ishighest on early haematopoietic progenitor cells and decreases as thecells mature. The antigen is absent on fully differentiatedhaematopoietic cells.

[0221] Uncommitted CD34⁺ progenitor cells are CD38⁺, DR⁻ and lacklineage-specific antigens, such as CD71, CD33, CD10, and CD5, whileCD34⁺ cells that are lineage-committed express the CD38 antigen in highdensity.

[0222] Most CD34⁺ cells reciprocally express either the CD45RO or CD45RAantigens. Approximately 60% of acute B-lymphoid leukaemia's and acutemyeloid leukaemia express the CD34 antigen. The antigen is not expressedon chronic lymphoid leukaemia (B or T lineage) or lymphomas. The CD2antigen is present on T lymphocytes and a subset of natural killerlymphocytes (NK).

[0223] The results are shown in Charts 2, 3 and 5.

[0224] Analysis of blood samples of a patient with B-CLL (Table 20,patient 5/6 at 2 hours) after treatment with monoclonal antibodies tothe β-chain of the DR antigen or the α-chain of the same antigenrevealed marked increases in the relative number of CD34⁺ and CD34⁺CD2⁺cells after treatment with the former antibody. Since the same bloodsamples were immunophenotyped with the above mentioned panels (seeTables 14 to 19) for other markers the increase in the relative numberof CD34⁺ and CD34 ⁺CD2⁺ cells observed here seems to coincide withincreases in the relative number of CD4⁺CD8⁺, CD8⁺CD3⁺ and CD4⁺CD3⁺single positive (SP) cells.

[0225] Furthermore, these findings which seem exclusive to engagement ofthe β-chain of the HLA-DR antigen, are in direct support that theprocess is giving rise to T-lymphopoiesis via B lymphocyte regression.

[0226] On analysing the same treatment 24 hours later the CD34⁺ cellsseemed to decrease in levels to give rise to further increase in therelative number of T lymphocytes. The process of retrodifferentiationthat initially gave rise to T-lymphopoiesis can be reversed to give riseto B-lymphopoiesis. The former phenomenon was observed at 2 hoursincubation time with monoclonal antibody to the β-chain of the HLA-DRantigen plus cylophosphoamide, whereas the latter process was noted at24 hours incubation time with the same treatment in the same sample(Chart 2).

[0227] Treatment of blood samples of HIV⁺ patient (Table 20 patientHIV⁺) with monoclonal antibody to the β-chain of the HLA-DR antigenmarkedly increased the relative number of CD34⁺ and CD2⁺CD34⁺ cells andso did treatment of the same blood sample with monoclonal antibody tothe β-chain of the HLA-DR antigen and monoclonal antibody to the α-chainof the same antigen when added together. However, treatment of thisblood sample with monoclonal antibody to the α-chain of the HLA-DRantigen did not affect the level of CD34⁺ cells. Treatment of bloodsamples obtained from a 6-day old baby (BB/ST Table 20) who wasinvestigated at that time for leukaemia and who had very high number ofatypical cells (blasts) in his blood with monoclonal antibody to theβ-chain of the HLA-DR antigen, or monoclonal antibody to the α-chain ofthe same antigen or both monoclonal antibodies added together resultedin the following immunophenotypic changes.

[0228] On analysis of untreated blood samples the relative number ofCD34⁺ and DR⁺ cells were markedly increased and on treatment withmonoclonal antibody to the β-chain the relative number of CD34⁺ cellsfurther increased but were noted to decrease on treatment withmonoclonal antibody to the β-chain of the HLA-DR antigen or treatmentwith monoclonal antibodies to the α and β-chains of the molecule whenadded together. However, the latter treatment increased the relativenumber of CD34 ⁺CD2⁺ cells and the opposite occurred when the same bloodsample was treated with monoclonal antibody to the β-chain of the HLA-DRantigen alone. On analysis of treated and untreated blood aliquots ofthe same patient 24 hours later the relative number of CD34⁺ decreasedwith all above mentioned treatments except it was maintained at a muchhigher level with monoclonal antibody to the β-chain of the HLA-DRantigen treatment. The latter treatment continued to decrease therelative number of CD34⁺CD2⁺ cells 24 hours later.

[0229] These results indicate that engagement of the HLA-DR antigen viathe β-chain promotes the production of more CD34⁺ cells from CD2⁺CD34⁺pool or from more mature types of cells such as B-lymphocytes ofpatients with B-CLL and these results indicate that this type oftreatment promotes retrodifferentiation. However, immunophenotyping ofblood samples 24 hours later suggests that these types of cells seem toexist in another lineage altogether and in this case cells seem to existor rather commit themselves to the myeloid lineage which was observed onanalysis of treated blood sample with the CD7 and CD13&33 panel.

[0230] Morphology changes immunophenotypic characteristics ofB-lymphocytes of B-CLL and enriched fractions of healthy individuals(using CD19 beads) on treatment with monoclonal antibodies to homologousregions of the β-chain of MHC class II antigens. These were accompaniedby a change in the morphology of B-lymphocytes. B-lymphocytes wereobserved colonising glass slides in untreated blood smears weresubstituted by granulocytes, monocytes, large numbers of primitivelooking cells and nucleated red blood cells. No mitotic figures orsignificant cell death were observed in treated or untreated bloodsmears.

[0231] The results of Table 20 also demonstrate a further importantfinding in that according to the method of the present invention it ispossible to prepare an undifferentiated cell by the retrodifferentiationof a more mature undifferentiated cell.

D. MICROSCOPE PICTUES

[0232] In addition to the antigen testing as mentioned above, the methodof the present invention was followed visually using a microscope.

[0233] In this regard, FIG. 1 is a microscope picture of differentiatedB cells before the method of the present invention. FIG. 2 is amicroscope picture of undifferentiated cells formed by theretrodifferentiation of the B cells in accordance with the presentinvention wherein the agent was a monoclonal antibody to the homologousregions of the β-chain of HLA-DR antigen. The undifferentiated cells arethe dark stained clumps of cells. FIG. 3 is a microscope picture of thesame undifferentiated cells but at a lower magnification.

[0234] FIGS. 1 to 3 therefore visually demonstrate theretrodifferentiation of B cells to undifferentiated stem cells by themethod of the present invention.

[0235]FIG. 4 is a microscope picture of differentiated B cells beforethe method of the present invention. FIG. 5 is a microscope picture ofundifferentiated cells formed by the retrodifferentiation of the B cellsin accordance with the present invention wherein the agent used was amonoclonal antibody to the homologous regions of the β-chain of HLA-DRantigen. Again, the undifferentiated cells are the dark stained clumpsof cells. FIG. 6 is a microscope picture of the formation ofdifferentiated granulocyte cells from the same undifferentiated cells ofFIG. 5.

[0236] FIGS. 4 to 6 therefore visually demonstrate theretrodifferentiation of B cells to undifferentiated stem cells by themethod of the present invention followed by commitment of theundifferentiated cells to new differentiated cells being of a differentlineage as the original differentiated cells.

[0237] The retrodifferentiation of T cells to undifferentiated stemcells by the method of the present invention followed by commitment ofthe undifferentiated cells to new differentiated cells being of adifferent lineage as the original differentiated cells was also followedby microscopy.

E. SUMMARY

[0238] In short, the examples describe in vitro experiments that revealextremely interesting findings regarding the ontogeny and development ofT and B lymphocytes which can be utilised in the generation of stemcells to affect lymphohaematopoiesis in peripheral blood samples in amatter of hours.

[0239] Treatment of peripheral blood samples obtained from patients withB-cell chronic lymphocytic leukaemia's (B-CLL) with high B lymphocytecounts, with monoclonal antibody to the homologous region of the β-chainof class-II antigens gave rise to a marked increase in the relativenumber of single positive (SP) T-lymphocytes and their progenitors whichwere double positive for the thymocyte markers CD4 and CD8 antigens andthese were coexpressed simultaneously. However, these phenomena werealways accompanied by a significant decrease in the relative number ofB-lymphocytes. These observations were not noted when the same bloodsamples were treated with monoclonal antibodies to the homologous regionof the α-chain of class-II antigens or to the homologous region ofclass-I antigens.

[0240] Treatment of whole blood obtained from patients with B-cellchronic lymphocytic leukaemia (CLL) with monoclonal antibody to thehomologous region of the B chain of the HLA-DR antigen appeared to giverise to T-lymphopoiesis. This event was marked by the appearance ofdouble positive cells coexpressing the CD4 and CD8 markers, theappearance of cells expressing CD34 and the concomitant increase in thenumber of single positive CD4⁺CD3⁺ and CD8⁺CD3⁺ lymphocytes.Furthermore, the immunophenotypic changes that took place in thegeneration of such cells were identical to those cited for thymocytedevelopment, especially when measured with time.

[0241] The percentages of double positive cells (DP) generated at 2 hourincubation time of whole blood with monoclonal antibody to thehomologous region of the β-chain of the DR antigen, decreased with timeand these events were accompanied by increase in the percentages ofsingle positive CD4⁺CD3⁺ and CD8⁺CD3 cells simultaneously and at latertimes too. TCR α and β chains were also expressed on these types ofcells.

[0242] B-lymphocytes were constantly observed to lose markers such asCD19, CD21, CD23, IgM and DR and this coincided with the appearance ofCD34⁺ and CD34⁺ CD2⁺ cells, increases in CD7⁺ cells, increases inCD8⁺CD28⁺ and CD28⁺cells, increases in CD25⁺ cells, the appearance ofCD10⁺ and CD34⁺ cells and CD34⁺ and CD19⁺ cells increases in CD5⁺ cells,and cells expressing low levels of CD45 antigen. These changes were dueto treatment of blood with monoclonal antibody to the homologous regionof the β-chain of HLA-DR antigen.

[0243] The immunophenotypic changes associated with such treatment isconsistent with retrodifferentiation and subsequent commitment (i.e.recommitment) of B lymphocytes, because the majority of white bloodcells in blood of patients with B-CLL before treatment were Blymphocytes. Furthermore, B-lymphocytes of patients with B-CLL whichwere induced to become T-lymphocytes following treatment withcyclophosphamide and monoclonal antibody to the β-chain of HLA-DRantigen, were able to revert back to B lymphocytes following prolongedincubation with this treatment.

[0244] On analysis of treated samples with monoclonal antibody to theβ-chain of HLA-DR antigen, with CD16&56 and CD3 and CD8 and CD3 panels,the relative number of cells expressing these markers steadily increasesin increments consistent with those determined with panels such as CD19and CD3 and DR and CD3. Investigation of the supernatant of treated anduntreated samples of patients with HIV infection using nephlometry andimmunoelectrophoresis reveals increased levels of IgG indicating thatthe B-cells must have passed through the plasma cell stage. The increasein the relative number of all above-mentioned cells was also accompaniedby the appearance of medium size heavily granulated cells expressing theCD56&16 antigens in extremely high amounts. Other cells which wereextremely large and heavily granulated were observed transiently andthese were positive for CD34 and double positive for CD4 CD8 markers.Other transient cells were also observed and these were large andgranular and positive for the CD3 and CD19 receptors. CD25 which waspresent on the majority of B-lymphocytes was lost and became expressedby newly formed T-lymphocytes which were always observed to increase innumber.

[0245] CD28⁺CD8⁺ and CD28⁺ cells appeared after treatment of whole bloodof patients with B-CLL with monoclonal antibody to the homologous regionof the B chain of the DR antigen. These findings were due to treatmentof blood with monoclonal antibody to the homologous region of theβ-chain of HLA-DR antigen. T-lymphopoiesis generated in this manner wasalso observed in peripheral blood of healthy blood donors, cord blood,bone marrow, patients with various infections including HIV⁺ individualsand AIDS patients, enriched fractions for B lymphocytes obtained fromblood samples of healthy blood donors, IgA deficient patients and otherpatients with various other conditions. Furthermore, analysis of myeloidmarkers in treated samples of two patients with B-CLL with monoclonalantibody to the homologous region of the Unchain of the HLA-DR antigenshowed a significant increase in the relative number of cells expressingthe myeloid markers such as CD13 and CD33. These markers werecoexpressed with the CD56 & 16 or the CD7 antigens. However, therelative number of CD7⁺ cells with T-lymphocyte markers and withoutmyeloid antigens was observed on a separate population of cells. Theseparticular observations were not seen in untreated samples or in samplestreated with monoclonal antibodies to class I antigens or the homologousregion of the α-chain of HLA-DR antigen (see Charts 2 & 3). These finalresults suggest that B-lymphocytes once triggered via the β-chain of theHLA-DR antigen are not only able to regress into T-lymphocyte progenitorcells but are also capable of existing into the myeloid and erythroidlineages.

[0246] It should be noted that the stem cells that are produced by themethod of the present invention may be stem cells of any tissue and arenot necessarily limited to lymphohaematopoietic progenitor cells.

[0247] Other modifications of the present invention will be apparent tothose skilled in the art. TABLE 1 CLINICAL DIAGNOSIS OF PATIENTS ANDEXPERIMENTAL CONDITIONS OF BLOOD SAMPLES INCLUDING COULTER COUNTS (WBC)FOLLOWING AND PRIOR TREATMENT OF BLOOD SPECIMENS WITH VARIOUS MONOCLONALANTIBODIES AND OTHER AGENTS WBC/L #LYMPH/L PATIENT EXPT X10-9 % LYMPH10X-9 AGENT ID DIAGNOSIS COND B A B A B A ML/mL 1 B-CLL 12 HR 100 ND86.1 ND 86.1 ND ANTI-B AT 22C. 50 2 B-CLL 2 HR 39.1 9.6 74.4 63.3 29.96.1 ANTI-B AT 22C. 50 2 HR AT 39.1 37.7 74.4 75.1 29.9 28.3 ANTI-B 22C.PE 50 3 B-CLL 6 HR 39.5 9.3 71.9 67.2 28.3 6.2 ANTI-B AT 22C. 50 6 HR39.5 37.7 71.9 72.5 28.3 27.4 ANTI-B AT 22C. PE 50 4 B-CLL 24 HR 39 9.373 66.5 28.4 6.2 ANTI-B AT 22C. 50 24 HR 39 36.2 73 70.4 28.4 25.5ANTI-B PE 50 5 B-CLL 2 HR ANTI-B AT 22C 50 ANTI-A 50 ANTI-I 50 ANTI-B&TOXIC AGENT 25 + 25 6 B-CLL 24 HR ANTI-B AT 22C. 50 7 B-CLL 24 HR 170128 95.4 91.1 16.9 11.6 ANTI-B AT 22C 10 178 94.2 16.8 ANTI-I 10 13090.4 11.9 ANTI-B & TOXIC AGENT 10 + 20 8 B-CLL 24 HR 16 7 81.9 51.2 143.0 ANTI-B AT 22C 20 9 B-CLL 12 HR +++ 89.5 87 85.1 +++ 76.2 ANTI-B AT22C. 30 +++ 85.4 +++ ANTI-I 30 89.4 ANTI-4 30 +++ 84.9 +++ ANTI- 95.4I + II + 4 10 + 10 + 10 10 B-CLL 2 HR 19.3 ND 86 ND 16.7 ND ANTI-B AT22C. 30 ANTI-I 30 92 OUT 2 HR 5.4 ND 74.5 ND ND ANTI-B PATIENT AT 22C.20 87 OUT 2 HR 4.8 ND 59.3 ND ND ANTI-B PATIENT AT 22C. 20 91 OUT 2 HR4.2 ND 54.0 ND ND ANTI-B PATIENT AT 22C. 20 21 OUT 2 HR 3.9 ND 47.4 NDND ANTI-B PATIENT AT 22C. 20 34 OUT 2 HR 7.2 ND 20.0 ND ND ANTI-BPATIENT AT 22C. 20 36 CMV 4 HR 13.4 ND 7.3 ND ND ANTI-B INFANT AT 22C.20 93 HIV + 4 HR 5.6 ND 43.4 ND ND ANTI-B INFANT AT 22C. 20 BB/ST 40%BLAST 2 HR AT 60.5 ND 20.2 ND 12.2 ND ANTI-B IN BLOOD 22C. 50 6 DAYS 24HR ANTI-A OLD AT 22C. 50 ANTI-AB 25 + 25 HIV25 AIDS 2 HR 7.5 ND 34.8 ND2.6 ND ANTI-B AT 22C. 50 ANTI-A 50 ANTI-AB 25 + 25 43/BD B CELL 4 HRANTI-B DEFICIENT AT 22C. 20 ANTI-I 20 ANTI-4 20 OB/BD B CELL 4 HR ANTI-BDEFICIENT AT 22C. 20 ANTI-I 20 ANTI-4 20 HIV+ AIDS 6 HR ANTI-B AT 22C.20 ANTI-I IgA-D IgA 6 HR ANTI-B DEFICIENT AT 22C. 20 ANTI-I 20

[0248] TABLE 2 IMMUNOPHENOTYPING OF PATIENTS WITH B-CLL AND OTHERCONDITIONS BEFORE AND AFTER TREATMENT OF BLOOD SAMPLES WITH MONOCLONALANTIBODY TO THE HOMOLOGOUS REGION OF THE B CHAIN OF THE HLA-DR WITH CD19AND CD3 MONOCLONAL ANTIBODIES. % CD3 − % CD19 + % CD19+ % CD3+ % CD19 +CD3+ CD19− HGCD3 − FC+ PATIENT B A B A B A B A B A 1 88 40 5 19 1 2 6 260 12 2 73 15 10 33 2 7 15 41 0 5 3 73 11 11 33 2 2 14 52 0 2 4 71 13 1137 2 2 16 47 0 2 5 85 40 5 16 1 1 6 26 3 18 6 85 43 5 18 1 1 6 27 3 10 790 72 2 4 0 2 7 8 0 14 8 62 25 7 13 0 1 29 55 2 6 9 90 85 2 3 0 0 2 1 14 10 78 50 7 14 0 0 14 26 0 8 92 12 10 38 49 0 1 49 40 0 0 91 7 3 35 290 1 59 67 0 0 87 5 3 32 38 1 1 63 58 0 0 21 1 1 27 29 1 0 71 70 0 0 34 11 13 13 0 2 86 84 0 0 39 10 6 23 25 0 0 67 69 0 0 93 6 3 26 27 1 1 68 700 0 BB/ST 1 1 12 13 0 0 87 86 0 0 HIV25 7 2 26 27 0 0 68 67 0 0 43/BD 00 40 42 0 1 58 54 0 0 04/BD 0 0 49 41 0 3 43 41 0 0 HIV+ 1 1 10 14 0 089 87 0 0 IgA/D 10 1 21 25 2 3 67 71 0 0

[0249] TABLE 3 IMMUNOPHENOTYPING OF PATIENTS WITH B-CLL AND OTHERCONDITIONS BEFORE AND AFTER TREATMENT OF BLOOD SAMPLES WITH MONOCLONALANTIBODY TO THE B CHAIN OF THE HOMOLOGOUS REGION OF THE HLA-DR WITHMONOCLONAL ANTIBODIES TO CD4 AND CD8. % CD8+ % CD4+ % CD4 + CD8+ % CD4 −CD8− CD4 + LOW PATIENT B A B A B A B A B A 1 28 16 2.9 11.4 0 3.2 93.167.6 0 0 2 62 13.2 9.1 24.3 0 9.4 78.7 46 5.8 6.3 3 7.2 13.1 7.4 23.9 08.2 78.8 48.1 6.3 6.6 4 10.1 24.2 7.6 24.9 0.3 2.8 77.5 42 4.6 5 5 2.916.2 1.8 7.6 0 2 95 62.3 0 0 6 ND 12 ND 8.1 ND 1.7 ND 75.7 ND 0 7 1.9 261.9 2.8 0 0 95.8 94.3 0 0 8 3.2 7 3.9 6.9 0.1 2 87.3 79.8 4.3 6 9 2.82.9 3 3 0 0 94 94.1 0 0 10 5.7 9.4 4.7 9.1 0.6 0.8 88.7 79.2 0 0 92 2119 21.6 21 0.8 1.9 50.5 52.5 5.3 4.8 91 15.4 18.1 13.6 17.9 6.2 2.6 5757.3 7.3 3.5 87 16.8 21.8 13.4 20.4 2.9 2.6 59.5 48.9 7 5.6 21 16 24.19.1 15.2 1 2.6 69.6 53.2 3.7 4.2 34 9.4 11.9 5.7 4.9 2 3.3 67.6 65.314.4 14.5 39 12.1 12.6 13.1 14.6 0.4 1.3 62.3 66.7 11.9 4.3 93 18.9 20.39.7 10.3 1.8 1.4 65.5 65.9 3.4 1.8 BB/ST 6.3 13 5.7 7.3 2.2 1.1 34.770.3 50.3 7.6 HIV25 24.1 24.9 0.8 1.1 1.3 5 70.2 69.3 2.9 3.8

[0250] TABLE 4 IMMUNOPHENOTYPING OF PATIENTS WITH B-CLL AND OTHERCONDITIONS BEFORE AND AFTER TREATMENT OF SAMPLES WITH MONOCLONALANTIBODY TO THE B CHAIN OF THE HLA-DR WITH MONOCLONAL ANTIBODIES TO CD3AND DR DR+ CD+ CD + DR+ DR − CD3− DR + HCD3− PATIENT B A B A B A B A B A1 87 45.5 3.5 20.8 2.5 4.2 6.9 21.6 0 7.6 2 76.2 19.4 9.6 29.2 3.9 8.710.3 36.8 0 5.5 3 77.7 18.3 8.4 29.4 4.1 8.8 9.6 38.1 0 4.7 4 76.8 19.27.6 29.5 6.2 10.5 9.1 37.2 0 3.3 5 ND 47.1 ND 11.5 ND 9.9 ND 22.4 ND 7.36 ND 7 91.4 85.8 2.4 2.5 0.7 0.7 5.1 4.2 0 6.3 8 61.0 28.9 6.5 11.2 23.3 28.6 54.6 0 1.5 9 ND 10 82.6 44.7 4.3 9.8 3.3 5 9.8 22.2 0 17.9 9223.8 14.1 39.3 41.9 4.5 3.5 32.4 40.5 0 0 91 13.3 7.9 29.6 32.5 3.4 2.953.4 56.5 0 0 07 14.8 12.2 28.4 34.1 5.5 6.6 51.1 46.5 0 0 21 ND 34 11.912.9 10.4 13.7 0.0 0.6 76.7 72.0 0 0 39 25.6 13.7 24.6 25.2 3 2.8 46.525.2 0 0 93 13.3 8.9 18.4 18.9 9.9 10.1 58.2 61.7 0 0 BB/ST 44.2 32.511.7 12.2 0.8 0.8 43 49.4 0 4.6

[0251] TABLE 5 IMNUNOPHENOTYPING OF PATIENTS WITH B-CLL AND OTHERCONDITIONS BEFORE AND AFTER TREATMENT OF BLOOD SAMPLES WITH MONOCLONALANTIBODY TO THE HOMOLOGOUS REGION OF THE B CHAIN OF THE HLA-DR WITHMONOCLONAL ANTIBODIES TO CD16 + 56 AND CD3. CD56+ CD56+ CD56+ & 16+ &16− & 16 CD3+ CD3+ CD3− PATIENTS B A B A B A B A 1 2 4.3 5.7 19.7 0.71.7 91.3 73 2 11.5 38.9 12.4 32.6 1 6.6 74.5 21 3 12 36.2 12.1 34.5 0.76 75.5 23 4 12.2 32.6 12.4 39.6 0.5 5 74.7 22.2 5 ND 13.1 ND 9.4 ND 2.6ND 73.5 6 ND 7 0.8 0.8 2.8 2.4 0.3 0.2 96.2 96.4 8 24.8 52 5.4 12.4 0.94.1 68.3 31.1 9 ND 10 1.1 1.3 6.1 13.7 2.1 2.5 90.5 82.4 92 23.8 34.544.3 44.8 2 1.5 29.2 18.6 91 4.6 3.9 28.8 29.4 3 3.2 63.3 63.3 87 47.946.4 28.8 36.5 5.8 3.7 16.9 13 21 9.4 9.4 19.7 23.6 4.2 6.7 66 59.5 3421.5 12.8 11.4 13.7 1.8 0.6 64.6 72.8 39 7 2.7 23.4 26.1 1.1 0.1 68.2 7193 55.8 54.9 26.2 26.3 1.7 2 16.1 16.8 BB/ST 28.8 29.9 12 14.3 0.8 1.849.4 53.6

[0252] TABLE 6 IMMUNOPHENTYPING OF PATIENTS WITH B-CLL AND OTHERCONDITIONS BEFORE AND AFTER OF TREATMENT OF BLOOD WITH MONOCLONALANTIBODY TO THE HOMOLOGOUS REGION OF THE B CHAIN OF THE HLA-DR WITHMONOCLONAL ANTIBODIES TO CD45 AND CD14. CD45+ CD45+H CD45+L CD14+PATIENTS B A B A B A 1 90.5 70.1 7.5 21.9 0.8 3.3 2 85.8 52.2 8.8 38.35.3 9.5 3 84.3 52.2 9.9 33.8 5.1 13.2 4 91.5 79.2 2.1 7 5.7 10.8 5 63.184.6 34.9 9.4 0.5 3.6 6 ND 7 52.8 85.2 45.6 13.9 0.5 0.6 8 71.1 55 71.134.5 5.3 8.7 9 SEE 10 79.7 47.3 16.3 48 2.1 1.9 92 61.7 64.7 27.4 26.65.9 3.6 91 49.4 49.2 40.4 44.3 6.5 3.2 87 52.4 61.5 36.1 28.7 7 6.5 2145.8 43.3 44.3 47.6 6.2 3.3 34 24.4 24.6 54.8 59.6 13.3 9.7 39 48.7 46.330.5 42.1 14.5 8.8 93 SEE HIV+ 22.6 26.9 66.8 63.5 6.8 6.7 IgA/D 47.459.8 41.9 33.3 5.9 4.1

[0253] TABLE 7 IMMUNOPHENOTYPING OF PATIENT WITH B-CLL AND OTHERCONDITIONS BEFORE AND AFTER TREATMENT OF BLOOD WITH MONOCLONALANTIBODIES TO THE HOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-DR WITHMONOCLONAL ANTIBODIES TO CD8 AND CD3. CD8+ CD8− CD8+ CD3+ CD3+ CD3−PATIENTS B A B A B A B A 2 0.6 1.3 7.5 19.3 4.2 19.3 87.7 63.8 3 1.1 1.48.3 20.3 5.6 18.4 84.8 59.8 4 3.5 2.9 8.3 27 3.9 16.6 84.2 53.1 92 3.51.9 27.6 25.2 18.4 19 50.3 52.8 91 4 3.1 18.2 19 14.1 12.6 63.6 65.3 875.7 3.9 19.9 23.6 15.4 17.4 58.8 55 21 4.8 7.4 16.3 17.3 13.7 13 65.2 6234 3 3.6 5.2 6.7 7.6 7.5 84.1 82.3

[0254] TABLE 8 IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITH TIME AFTERTREATMENT OF BLOOD WITH PE CONJUGATED MONOCLONAL ANTIBODY TO THEHOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-DR MEASURE WITH MONOCLONALANTIBODIES TO CD45 AND CD14. TIME DR+CD45+CD14+r CD45+L CD45+H  2 HR81.7 8.2 0.2  6 HR 80.7 8.1 10.6 24 HR 79 1.1 18.4

[0255] TABLE 9 IMMUNOPHENOTYPING OF A PATINENT WITH B-CLL WITH TIMEAFTER TREATMENT OF BLOOD WITH PE CONJUGATED MONOCLONAL ANTIBODY TO THEHOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-DR MEASURED WITH MONOCLONALANTIBODIES TO CD19 AND CD3. CD19− TIME CD19+DR+r CD3+ CD3+DR+ CD3−DR−  2HR 87.4 10.1 1.8 10.7  6 HR 75.5 10.4 3.1 10.7 24 HR 74 11.7 2.9 11

[0256] TABLE 10 IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITH TIMEAFTER TREATMENT OF BLOOD WITH PE CONJUGATED MONOCLONAL ANTIBODY TO THEHOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-DR MEASURED WITH MONOCLONALANTIBODIES TO CD4 AND CD8. TIME CD8+ & DR + r CD4+ CD4+ & CD8+& DR + rCD4 + DR+ CD4 − CD8 − DR− 2 HR 77.6 6.8 5.4 1.3 8.8 6 HR 75.8 6.7 64 1.89.3 24 HR 77 6.4 48 1.9 11

[0257] TABLE 11 IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITH TIMEAFTER TREATMENT OF BLOOD WITH PE CONJUGATED MONOCLONAL ANTIBODY TO THEHOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-DR MEASURED WITH MONOCLONALANTIBODIES TO CD3 AND DR. TIME DR+ CD3+ CD3+DR+ CD3+DR−  2 HR 75 9.5 4.210.9  6 HR 74.8 8.8 4.8 10.9 24 HR ND ND ND ND

[0258] TABLE 12 IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITH TIMEAFTER TREATMENT OF BLOOD WITH PE CONJUGATED MONOCLONAL ANTIBODY TO THEHOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-DR MEASURED WITH MONOCLONALANTIBODIES TO CD16 & 56 AND CD3. CD56+ & CD56+CD16+ CD56−CD16− TIME16+DR+r CD3+ & CD3+DR+r & CD16−DR−  2 HR 82.5 9.5 4.1 3.5  6 HR 84.3 7.54.1 3.3 24 HR ND ND ND ND

[0259] TABLE 13 IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITH TIMEAFTER TREATMENT OF BLOOD WITH PE CONJUGATED MONOCLONAL ANTIBODY TO THEHOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-DR MEASURED WITH MONOCLONALANTIBODIES TO CD8 AND CD3. CD8 + CD + TIME CD8 + DR + CD3 + 3 & DR + rCD8 − CD3 − DR − 2 HR 76.2 6.6 6.7 10.6 6 HR 76.5 6.2 6.2 10.3

[0260] TABLE 14 IMMUNOPHENOTYPING OF PATIENTS WITH B-CLL BEFORE ANDAFTER TREATMENT OF BLOOD WITH MONOCLONAL ANTIBODIES TO THE HOMOLOGOUSREGION OF THE A-CHAIN OF THE HLA-DR, THE HOMOLOGOUS REGION OF THEB-CHAIN OF THE HLA-DR. THE TWO MONOCLONAL TOGETHER. MONOCLONAL TO THEHOMOLOGOUS REGION OF THE B-CHAIN PLUS CYCLOPHOSPHOAMIDE AND THEHOMOLOGOUS REGION OF CLASS I ANTIGENS MEASURED WITH TIME. CD19+ CD3+CD19+CD3+ CD19−CD3− A A A A A A A A ID B A AB BC AI B A AB BC AI B A ABBC AI B A AB BC AI 5/ 6 2H 86 91 54 40 89 5 4 16 23 5 1 1 3 2 1 6 4 2733 5 24 N 88 51 60 86 N 4 18 10 4 N 2 1 2 3 N 4 29 28 7 10 2H 77 N 59 N80 7 N 13 N 7 1 N 1 N 0 14 N 26 N 12 09 24 8 N N N 6 32 N N N 38 1 N N N1 59 N N N 56 43 /B D 6H 0 N 0 0 0 40 N 42 43 49 0 N 1 0 1 58 N 54 54 4704 /B D 6H 0 N 0 0 0 49 N 41 45 46 0 N 3 1 3 43 N 42 44 41 HI V + 6H 1 N0 N 1 10 N 14 N 12 0 N 0 N 0 89 N 86 N 87 Ig A/ D 6H 10 N 1 N 12 21 N 25N 20 2 N 1 N 3 67 N 71 N 68

[0261] TABLE 15 CD8 AND CD4 CDB+ CD4+ CD4+CD8+ CD4−CD8− A A A A A A A AID B A AB BC AI B A AB BC AI B A AB BC AI B A AB BC AI 5/ 6 2H 3 2 14 104 2 2 8 8 3 0 0 3 2 1 95 94 74 79 93 24 N 3 9 4 4 N 3 8 4 3 N 0 2 2 0 N94 81 90 93 10 2H 3 N 7 N 4 4 N 7 N 3 1 N 2 N 1 91 N 83 N 92 09 24 10 NN N 15 21 N N N 38 2 N N N 2 61 N N N 53

[0262] TABLE 16 CD3 AND DR DR+ CD3+ CD3+DR+ CD3−DR− A A A A A A A A ID BA AB BC AI B A AB BC AI B A AB BC AI B A AB BC AI 5/ 6 2H N 90 54 N 87 N4 12 N 4 N 2 10 N 3 N 5 22 N  5 10 2H 83 N 63 N 81  4 N  8 N  4 4 N  7 N4  9 N 23 N 12 09 24 14 N N N 13 30 N N N 36 3 N N N 3 51 N N N 47

[0263] TABLE 17 CD16 & 56 AND CD3 CD56+ & CD56− & CD56+ & 16+ CD3+16+CD3+ 16 − CD3 − A A A A A A A A ID B A AB BC AI B A AB BC AI B A ABBC AI B A AB BC AI 5/ 6 2H N 0 13 N 4 N 5  9 N  5 N 1 3 N 1 N 94 74 N 9010 2H  0 N  1 N 1  6 N 14 N  6 1 N 2 N 1 92 N 65 N 92 09 24 42 N N N 4136 N N N 38 2 N N N 2 20 N N N 19

[0264] TABLE 18 CD45 AND CD14 CD45+L CD45+M CD45+H CD45 + CD14 + A A A AA A A A ID B A AB BC AI B A AB BC AI B A AB BC AI B A AB BC AI 5/ 6 2H 00 5 10 0 44 43 50 50 32 55 43 50 31 67 1 1 1 2 0 10 2H 0 N 0 N 0 43 N 54N 35 54 N 42 N 62 1 N 1 N 0 09 24 2 N N N 1 18 N N N 16 71 N N N 76 7 NN N 5 HI V+ 6H 4 N 3 N 6 63 N 61 N 41 23 N 27 N 40 7 N 7 N 7 Ig A/ D 6H2 N 2 N 4 40 N 31 N 44 47 N 60 N 44 6 N 4 N 6

[0265] TABLE 19 CD8 AND CD28 CD8+ CD28+ CD8+CD28+ CD8−CD28− A A A A A AA A ID B A AB BC AI B A AB BC AI B A AB BC AI B A AB BC AI 5/ 6 2H N 3 6N 3 N 1 4 N 2 N 1 4 N 1 N 95 86 N 94 B 2H 4 N 6 N N 3 N 5 N N 1 N 3 N N9 N 86 N N 2

[0266] TABLE 20 CD34 AND CD2 CD34+ CD2+ CD34+CD2+ CD34−CD2− A A A A A AA A A ID B A AB BC I B A AB BC AI B A AB BC AI B A AB BC AI 5/ 6 2H N  134 N N N  6 13 N N N  3 30 N N N 90 21 N N 24 N  1  6  9 N N  7 23  4 NN  3 33 43 N N 87 34 34 N HI V+ 2H  2  1 12 13 N 20 21 21 12 N 4  5  914 N 7 73 64 60 N 3 BB /S T 2H 26 23 33 14 N 15 14 15 15 N 3 30 23 36 N2 32 28 35 N 1 7 24 N 11 29 11 N N 13 12  9 N N 27  9 18 N N 48 49 61 N

[0267] CHART 1 IMMUNOPHENOTYPIC CHANGES OF UNTREATED AND TREATED BLOODSAMPLE OF PATIENT (2, 3 & 4) WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUSREGION OF THE β-CHAIN OF HLA-DR ANTIGEN MEASURED WITH TIME. WITHOUT WITHFL1 FL2 TIME NOTHING001 WITH002 CD45 CD14 2 HR NO001 WE002 CD45 CD14 6HR 001001 002002 CD45 CD14 24 HR NOTHING003 WITH004 CD3 CD19 2 HR NO003WE004 CD3 CD19 6 HR 001003 002004 CD3 CD19 24 HR NOTHING004 WITH005 CD4CD8 2 HR NO004 WE005 CD4 CD8 6 HR 001004 002005 CD4 CD8 24 HR NOTHING005WITH006 CD3 DR 2 HR NO005 WE006 CD3 DR 6 HR 00I005 002006 CD3 DR 24 HRNOTHING006 WITH007 CD3 CD56 & 16 2 HR NO006 WE007 CD3 CD56 & 16 6 HR001006 002007 CD3 CD56 & 16 24 HR N003 W004 CD3 CD8 2 HR NO007 WE008 CD3CD8 6 HR 001007 002008 CD3 CD8 24 HR

[0268] CHART 1A IMMUNOPHENOTYPIC CHANGES OF UNTREATED AND TREATED BLOODSAMPLE OF PATIENT (2, 3, 4) WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUSREGION OF THE β-CHAIN OF HLA-DR ANTIGEN CONJUGATED TO PE MEASURED WITHTIME. ID FL1 FL2 TIME WL003 CD45 CD14 2 HR WEL003 CD45 CD14 6 HR 003003CD45 CD14 24 HR WL005 CD3 CD19 2 HR WEL005 CD3 CD19 6 HR 003005 CD3 CD1924 HR WL006 CD4 CD8 2 HR WEL006 CD4 CD8 6 HR 003006 CD4 CD8 24 HR WL007CD3 DR 2 HR WEL 007 CD3 DR 6 HR WL008 CD3 CD65 & 16 2 HR WEL 008 CD3CD56 & 16 6 HR WL005 CD3 CD8 2 HR WEL009 CD3 CD8 6 HR

[0269] CHART 2 IMMUNOPHENOTYPIC CHANGES OF UNTREATED AND TREATED BLOODOF PATIENT (1) WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THEβ-CHAIN OF HLA-DR ANTIGEN. THIS ANTIBODY AND CYCLOPHOSPHANIDE,MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE α-CHAIN OF HLA-DRANTIGEN AND MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF CLASS IANTIGEN MEASURED WITH TIME. WITH WITHOUT FL1 FL2 TIME NA001 CD45 CD14 2HR A2B001: AB CD45 CD14 2 HR A2A: AA CD45 CD14 2 HR DNAA001: ABC CD45CD14 2 HR A1001: AI CD45 CD14 2 HR NC001 CD3 CD19 2 HR C2B001.AB CD3CD19 2 HR C2A001: AA CD3 CD19 2 HR DNAC001.ABC CD3 CD19 2 HR C1001: AICD3 CD19 2 HR A124H001: AI CD3 CD19 24 HR A2B24H001: AB CD3 CD19 24 HRA2A24H001: AA CD3 CD19 24 HR A2BX24H001: ABC CD3 CD19 24 HR ND001 CD4CD8 2 HR D2B001: AB CD4 CD8 2 HR D2A001: AA CD4 CD8 2 HR DNAD001.ABC CD4CD8 2 HR D1001: AI CD4 CD8 2 HR D124H001: AI CD4 CD8 24 HR D2BX24H001:ABC CD4 CD8 24 HR D2B001: AB CD4 CD8 24 HR D2A001: AA CD4 CD8 24 HRE1001: AI CD3 DR 2 HR E2B001: AB CD3 DR 2 HR E2A001: AA CD3 DR 2 HRF1001: AI CD3 CD56 & 16 2 HR F2B001: AB CD3 CD56 & 16 2 HR F2A001: AACD3 CD56 & 16 2 HR G1001: AI CD28 CD8 2 HR G2A001: AA CD28 CD8 2 HRG2B001: AB CD28 CD8 2 HR H1001: AI CD7 CD33 & 13 2 HR H2A001: AA CD7CD33 & 13 2 HR H2B001: AB CD7 CD33 & 13 2 HR I2A001: AA CD21 CD5 2 HRI2B001: AB CD21 CD5 2 HR J2A001: AA CD34 CD2 2 HR J2B001. AB CD34 CD2 2HR B2A24H001: AA CD34 CD2 24 HR B2824H001: AB CD34 CD2 24 HR B2BX24H001:ABC CD34 CD2 24 HR K2B001: AB CD10 CD25 2 HR K2A001: AA CD10 CD25 2 HR

[0270] CHART 3 IMMUNOPHENOTYPIC CHANGES OF UNTREATED AND TREATED BLOODOF PATIENT (8) WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THEβ-CHAIN OF HLA-OR ANTIGEN. WITH WITHOUT FL1 FL2 TIME AN001 CD45 CD14 2HR A2001 CD45 CD14 2 HR CN001 CD3 CD19 2 HR C2001 CD3 CD19 2 HR DN001CD4 CD8 2 HR D2001 CD4 CD8 2 HR EN001 CD3 DR 2 HR E2001 CD3 DR 2 HRFN001 CD3 CD56 & 16 2 HR F2001 CD3 CD56 & 16 2 HR GN001 CD28 CD8 2 HRG2001 CD28 CD8 2 HR HN001 CD7 CD5 2 HR H2001 CD7 CD5 2 HR IN001 CD13CD20 2 HR I2001 CD13 CD20 2 HR JN001 CD45RA CD25 2 HR J2001 CD45RA CD252 HR KN001 CD57 CD23 2 HR K2001 CD57 CD23 2 HR

[0271] CHART 4 IMMUNOPHENOTYPIC CHANGES OF UNTREATED AND TREATED BLOODSAMPLE OF PATIENT (10) WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGIONOF THE β-CHAIN OF HLA-DR ANTIGEN AND MONOCLONAL ANTIBODY TO THEHOMOLOGOUS REGION OF CLASS I ANTIGENS. WITH WITHOUT FL1 FL2 TIME CLL0001CD45 CD14 2 HR CLL1001 CD45 CD14 2 HR CLL2001 CD45 CD14 2 HR CLL0003 CD3CD19 2 HR CD3 CD19 2 HR CLL1003 CD3 CD19 2 HR CLL2003 CD3 CD19 2 HRCLL0004 CD4 CD8 2 HR CLL1004 CD4 CD8 2 HR CLL2004 CD4 CD8 2 HR CLL005CD3 DR 2 HR CLL1005 CD3 DR 2 HR CLL2005 CD3 DR 2 HR CLL0006 CD3 CD56 &16 2 HR CLL1006 CD3 CD56 & 16 2 HR CLL2006 CD3 C056 & 16 2 HR

1. A method of preparing an undifferentiated cell, the method comprisingcontacting a more committed cell with an agent that causes the morecommitted cell to retrodifferentiate into an undifferentiated cell.
 2. Amethod according to claim 1 wherein the more committed cell is capableof retrodifferentiating into an MHC Class I⁺ and/or an MHC Class II⁺undifferentiated cell.
 3. A method according to claim 1 or claim 2wherein the more committed cell is capable of retrodifferentiating intoan undifferentiated cell comprising a stem cell antigen.
 4. A methodaccording to any one of the preceding claims wherein the more committedcell is capable of retrodifferentiating into a CD34⁺ undifferentiatedcell.
 5. A method according to any one of the preceding claims whereinthe more committed cell is capable of retrodifferentiating into alymphohaematopoietic progenitor cell.
 6. A method according to any oneof the preceding claims wherein the more committed cell is capoable ofretrodifferentiating into a pluripotent stem cell.
 7. A method accordingto any one of the preceding claims wherein the undifferentiated cell isan MHC Class I⁺ and/or an MHC Class II⁺ cell.
 8. A method according toany one of the preceding claims wherein the undifferentiated cellcomprises a stem cell antigen.
 9. A method according to any one of thepreceding claims wherein the undifferentiated cell is a CD34⁺undifferentiated cell.
 10. A method according to any one of thepreceding claims wherein the undifferentiated cell is alymphohaematopoietic progenitor cell.
 11. A method according to any oneof the preceding claims wherein the undifferentiated cell is apluripotent stem cell.
 12. A method according to any one of thepreceding claims wherein the more committed cell is an MHC Class I⁺ andor an MHC Class II⁺ cell.
 13. A method according to any one of thepreceding claims wherein the agent acts extracelluarly of the morecommitted cell.
 14. A method according to any one of the precedingclaims wherein the more committed cell comprises a receptor that isoperably engageable by the agent and wherein the agent operably engagesthe receptor.
 15. A method according to claim 14 wherein the receptor isa cell surface receptor.
 16. A method according to claim 14 or claim 15wherein the receptor comprises an α-component and/or a β-component. 17.A method according to claim 16 wherein the receptor comprises a β-chainhaving homologous regions.
 18. A method according to claim 17 whereinthe receptor comprises at least the homologous regions of the β-chain ofHLA-DR.
 19. A method according to claim 16 wherein the receptorcomprises an α-chain having homologous regions.
 20. A method accordingto claim 19 wherein the receptor comprises at least the homologousregions of the α-chain of HLA-DR.
 21. A method according to any one ofclaims 14 to 20 wherein the agent is an antibody to the receptor.
 22. Amethod according to claim 21 wherein the agent is a monoclonal antibodyto the receptor.
 23. A method according to claim 18 wherein the agent isan antibody, preferably a monoclonal antibody, to the homologous regionsof the β-chain of HLA-DR.
 24. A method according to claim 20 wherein theagent is an antibody, preferably a monoclonal antibody, to thehomologous regions of the α-chain of HLA-DR.
 25. A method according toany one of the preceding claims wherein the agent modulates MHC geneexpression, preferably wherein the agent modulates MHC Class I⁺ and/orMHC Class II⁺ expression.
 26. A method according to any one of thepreceding claims wherein the agent is used in conjunction with abiological response modifier.
 27. A method according to claim 25 whereinthe biological response modifier is an alkylating agent, preferablywherein the alkylating agent is or comprises cyclophosphoamide.
 28. Amethod according to any one of the preceding claims wherein the morecommitted cell is a differentiated cell.
 29. A method according to claim28 wherein the more committed cell is any one of a B cell or a T cell.30. A method according to any one of claims 1 to 27 wherein the morecommitted cell is a more mature undifferentiated cell.
 31. A methodaccording to any one of the preceding claims wherein theundifferentiated cell is committed to a recommitted cell.
 32. A methodaccording to claim 31 wherein the recommitted cell is of the samelineage as the more committed cell prior to retrodifferentiation.
 33. Amethod according to claim 31 wherein the recommitted cell is of adifferent lineage as the more committed cell prior toretrodifferentiation.
 34. A method according to any one of claims 31 to33 wherein the recommitted cell is any one of a B cell, a T cell or agranulocyte.
 35. A method according to any one of the preceding claimswherein the method is an in vitro method.
 36. An undifferentiated cellproduced according to the method of any one of claims 1 to
 35. 37. Anundifferentiated cell produced according to the method of any one ofclaims 1 to 35 for use as or in the preparation of a medicament.
 38. Useof an undifferentiated cell produced according to the method of any oneof claims 1 to 37 in the manufacture of a medicament for the treatmentof an immunological disorder or disease.
 39. A recommitted cell producedaccording to the method of any one of claims 31 to
 35. 40. A recommittedcell produced according to the method of any one of claims 31 to 35 foruse as or in the preparation of a medicament.
 41. Use of a recommittedcell produced according to the method of any one of claims 31 to 35 inthe manufacture of a medicament for the treatment of an immunologicaldisorder or disease.
 42. A more committed cell having attached theretoan agent that can cause the more committed cell to retrodifferentiateinto an undifferentiated cell.
 43. A CD19⁺ and CD3⁺ cell.
 44. A methodof preparing an undifferentiated cell from a more committed cellsubstantially as hereinbefore described.
 45. An undifferentiated cellprepared from a more committed cell substantially as hereinbeforedescribed.
 46. A recommitted cell prepared from an undifferentiated cellwhich has been prepared from a more committed cell substantially ashereinbefore described.